Cell-based therapy for the pulmonary system

ABSTRACT

Cell based therapy comprises administration to the lung by injection into the blood system of viable, mammalian cells effective for alleviating or inhibiting pulmonary disorders. The cells may express a therapeutic transgene or the cells may be therapeutic in their own right by inducing regenerative effects.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/236,980 filed Sep. 9, 2002 currently pending, which is acontinuation-in-part of U.S. patent application Ser. No. 09/404,652filed Sep. 24, 1999 and issued as U.S. Pat. No. 6,482,406, which is acontinuation-in-part of U.S. patent application Ser. No. 09/276,654filed Mar. 26, 1999 and now issued as U.S. Pat. No. 6,592,868. Theentire disclosure of those applications is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to medical treatments and composition andprocedures useful therein. More specifically, it relates to cell-basedtherapy delivered to the pulmonary system of a mammalian patient.

BACKGROUND OF THE INVENTION

Cell-based gene transfer is a known, albeit relatively new andexperimental, technique for conducting gene therapy on a patient. Inthis procedure, DNA sequences containing the genes which it is desiredto introduce into the patient's body (the transgenes) are preparedextracellularly, e.g. by using enzymatic cleavage and subsequentrecombination of DNA with insert DNA sequences. Mammalian cells such asthe patient's own (i.e. autologous) or cells from another individual(i.e. allogenic) cells are then cultured in vitro and treated so as totake up the transgene in an expressible form. The transgenes may beforeign to the mammalian cell, additional copies of genes alreadypresent in the cell, to increase the amount of expression product of thegene or copies of normal genes which may be defective or missing in aparticular patient. Then the cells containing the transgene areintroduced into the patient, so that the gene may express the requiredgene products in the body, for therapeutic purposes. The take-up of theforeign gene by the cells in culture may be accomplished by geneticengineering techniques, e.g. by causing transfection of the cells with avirus containing the DNA of the gene to be transferred by lipofection,by electroporation, or by other accepted means to obtain transfectedcells, such as the use of viral vectors. This is sometimes followed byselective culturing of the cells which have successfully taken up thetransgene in an expressible form, so that administration of the cells tothe patient can be limited to the transfected cells expressing thetransgene. In other cases, all of the cells subject to the take-upprocess are administered.

This procedure has in the past required administration of the cellscontaining the transgene directly to the body organ requiring treatmentwith the expression product of the transgene. Thus, transfected cells inan appropriate medium have been directly injected into the liver or intothe muscle requiring the treatment, or via the systemic arterialcirculation to enter the organ requiring treatment.

Previous attempts to introduce such genetically modified cells into thesystemic arterial circulation of a patient have encountered a number ofproblems. For example, there is difficulty in ensuring a sufficientlyhigh assimilation of the genetically modified cells by the specificorgan or body part where the gene expression product is required forbest therapeutic benefit. This lack of specificity leads to theadministration of excessive amounts of the genetically modified cells,which is not only wasteful and expensive, but also increases risks ofside effects. In addition, many of the transplanted genetically modifiedcells do not survive when administered to the systemic arterialcirculation, since they encounter relatively high arterial pressures.Infusion of particulate materials, including cells, to other systemiccirculations such as the brain and the heart, may lead to adverseconsequences due to embolization, i.e. ischemia and even infarction.

The acute respiratory distress syndrome (ARDS), the clinical correlateof severe acute lung injury (ALI) in humans, is an important cause ofmorbidity and mortality in critically ill patients (1-4). Infectiousetiologies, such as sepsis and pneumonia (including influenza and SARS),are leading causes of ALI/ARDS (1, 2). Histologically, ALI/ARDS inhumans is characterized by a severe acute inflammatory response in thelungs and neutrophilic alveolitis (1). Inflammatory stimuli frommicrobial pathogens, such as endotoxin (lipopolysaccharide, LPS), arewell recognized for their ability to induce pulmonary inflammation, andexperimental administration of LPS, both systemically andintratracheally, has been used to induce pulmonary inflammation inanimal models of ALI (5-9).

The physiological hallmark of ARDS is disruption of thealveolar-capillary membrane barrier (i.e., pulmonary vascular leak),leading to development of non-cardiogenic pulmonary edema in which aproteinaceous exudate floods the alveolar spaces, impairs gas exchange,and precipitates respiratory failure (1, 10, 11). Both alveolarepithelial and endothelial cell injury and/or death have been implicatedin the pathogenesis of ALI/ARDS (1). However, despite decades ofresearch, few therapeutic strategies for clinical ARDS have emerged andcurrent specific options for treatment are limited (12-16). ARDScontinues to be an important contributor to prolonged mechanicalventilation in the intensive care unit (ICU), and ARDS-associatedmortality remains high at 30-50% despite optimal ICU supportive care (1,13, 14, 16).

ARDS is a complex clinical syndrome which is initiated by injury to thelung, often in the setting of pneumonia and/or sepsis, and aggravated byventilator-induced injury. Some of the early feature of ARDS can bereproduced by administration of bacterial endotoxin (LPS), which actsvia Toll-like receptor 4 (TLR4), to increase the expression ofinflammatory cyitokines and chemokines, and upregulate leukocyteadhesion molecules, results in EC activation (5-9, 57).

It is an object of the present invention to provide a novel procedure ofcell based therapy or cell-based gene transfer to mammals, for thetreatment of lung diseases or disorders.

It is a further and more specific object of the invention to providenovel procedures of cell-based gene therapy utilizing dermal (or other)fibroblast cells, EPCs, or MSCs, for treatment of lung diseases ordisorders.

It is a further object of the invention to provide novel geneticallyengineered cells containing transgenes expressing angiogenic factors fortreatment of lung diseases or disorders.

It is a further and more specific object of the invention to providenovel uses and novel means of administration of angiogenic factors inhuman patients for treatment of lung diseases or disorders.

It is a further object of the invention to treat or prevent pulmonaryhypertension utilizing novel therapies, including cell therapy andcell-based gene therapy.

It is a further object of the present invention to treat or preventAcute Respiratory Distress Syndrome (ARDS) utilizing novel therapies,including cell therapy and cell-based gene therapy.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery that the pulmonarysystem of a mammal, including a human, offers a potentially attractivemeans of introducing genetically altered cells or regenerative cellsinto the body, for purposes of gene therapy, i.e. cell based genetransfer, or for pulmonary regeneration cell therapy. The pulmonarysystem has a number of unique features rendering it particularly suitedto a cell-based gene transfer. Thus, low arterial pressure and highsurface area with relatively low shear in the micro-circulation of thelungs increase the chances of survival of the transplanted cells. Highoxygenation in the micro-circulation of the ventilated lung alsoimproves the viability of the transplanted cells.

Moreover, the pulmonary circulation functions as a natural filter, andis able to retain the infused cells efficiently and effectively. Also,the lung has a dual circulation (pulmonary arterial and bronchial). Thisis in contra-distinction to other systemic circulations, such as thebrain and the heart, where the infusion of particulate materials such ascells could lead to the aforementioned adverse consequences. The lungpresents a massive vascular system. The high surface area of thepulmonary endothelium allows the migration of the transplanted cellstrapped in the micro-circulation across the endothelial layer to take upresidence within the perivascular space.

The pulmonary circulation, unlike any other circulation in the body,receives the entire output of the heart. Accordingly, it offers thegreatest opportunity to release a gene product into the circulation.This distinct property of the lung is particularly useful for pulmonarygene therapy and for the treatment of a systemic disorders, as well as apulmonary disorder.

It is believed that the cells become lodged in the smallartery-capillary transition regions of the pulmonary circulation system,following simple intravenous injection of the transfected orregenerative cells to the patient. Products administered intravenouslymove with the venous circulation to the right side of the heart and thento the lungs. The cells administered according to the invention appearto lodge in the small arteriolar-capillary transition regions of thecirculatory system of the lungs, and then transmigrate from theintraluminal to the perivascular space. From there transfected cells candeliver expression products of the transgenes to the lungs, making theprocess to the present invention especially applicable to treatment ofpulmonary disorders. Some factors, especially stable factors can besecreted to the general circulation for treatment of disorders of otherbody organs.

Certain cells may have therapeutic potential in their own right, such asbone marrow derived (mesenchymal) stem (stromal) cells (MSCs) or othercells with regenerative potential (e.g. endothelial progenitor cells orendothelial-like progenitor cells, adipose tissue derived mesenchymalstem cells, multipotent adult progenitor cells (MAPCs), side population(SP) cells, lung derived progenitor or stem cells, or embryonic stemscells (ESCs), among others) in which case administration of such cellseven without the benefit of gene transfection may result in therapeuticeffects.

Thus, according to a first aspect of the present invention, there isprovided a process of conducting gene therapy in a mammalian patient,which comprises administering to the pulmonary system of the patient,genetically modified mammalian cells containing at least one expressibletransgene which is capable of producing at least one gene product in thepulmonary circulation after administration thereto.

According to another, more specific aspect of the invention, there areprovided genetically modified mammalian cells selected from fibroblasts,endothelial cells, smooth muscle cells, endothelial progenitor cells,endothelial-like progenitor cells, and mesenchymal stem cells, saidcells containing at least one expressible transgene coding for atherapeutic factor.

A further aspect of the present invention provides the use in thepreparation of a medicament for administration to a mammalian patient toalleviate symptoms of a disorder, of viable, transfected mammalian cellscontaining at least one expressible transgene coding for a therapeuticfactor.

Yet another aspect of the present invention is a process of preparinggenetic modifications of mammalian cells selected from fibroblasts,endothelial cells and progenitor cells, which comprises transfectingsaid mammalian cells with at least one gene coding for a therapeuticfactor, to produce transfected cells capable of expressing saidtherapeutic factor in vivo.

An additional aspect of the present invention is the treatment ofpulmonary hypertension (PH). Primary pulmonary hypertension (PPH), nowreferred to as idiopathic PAH (IPAH), and other causes of PH areassociated with severe abnormalities in endothelial function, whichlikely play a critical role in its pathogenesis. The vasodilatory,anti-thrombotic and anti-proliferative factor, nitric oxide (NO) hasbeen demonstrated to decrease pulmonary pressures in both experimentaland clinical situations. However, long-term viral-based methods maycause significant local inflammation. Other, previous attempts to treatPPH have involved the use of prostacyclin, using continuousadministration, but this is a difficult and expensive procedure, liableto give rise to side effects. Newer oral, inhaled or subcutaneouslyadministered treatments have been recently introduced, but, again, thesehave limited efficacy and/or significant side effects which limit theiruse.

The present invention provides, from this additional aspect, a method ofalleviating the symptoms of IPAH (and other causes of PH) whichcomprises administering to the pulmonary system of a patient sufferingtherefrom, at least one angiogenic factor, or a precursor or geneticproduct capable of producing and releasing into the pulmonarycirculation at least one angiogenic factor. The method would beapplicable to all “Group 1” WHO PAH including PAH associated withscleroderma, congenital heart disease, lupus (SLE), etc.

An embodiment of this additional aspect of the present invention is thedelivery to a patient suffering from PPH of genetically modified cellscontaining a gene capable of expressing in vivo at least one angiogenicfactor, by a process of cell-based gene transfer as described above.This additional aspect of invention, however, is not limited to anyspecific form of administration, but pertains generally to the use ofangiogenic factors and precursors thereof which produce angiogenicfactors in situ, in treating or alleviating the symptoms of PPH,delivered to the pulmonary circulation by any suitable means.

The invention provides a process of alleviating or inhibiting a disorderin a mammalian patient by conducting therapy which comprisesadministration to the lung by injection into the blood system of themammalian patient suffering from a disorder, of viable mammalian cellseffective for alleviating or inhibiting the disorder.

The mammalian cells may contain at least one expressed transgene, thetransgene expressing a composition effective for alleviating orinhibiting the disorder.

In an embodiment, the disorder is a breathing disorder. Breathingdisorders may be due to disorders of the lung or airways. In anembodiment, the transfected cells contain a transgene coding forProstaglandin I Synthase (PGIS). The breathing disorder may be ARDS. Thetransfected cells may contain a transgene coding for Ang-1. The disordermay be cystic fibrosis. The transfected cells may contain a transgenecoding for CFTR.

One aspect of the present invention is the treatment or prevention ofARDS through the administration of MSC cells. The MSC cells may betransfected or otherwise transformed to express Ang-1.

The invention further teaches genetically modified, viable cellsgenetically modified to contain an expressible transgene coding forPGIS. The cells may be fibroblasts. The cells may be for use in thetreatment of pulmonary hypertension. The cells may be for use in thetreatment of PPH.

The invention further teaches genetically modified, viable cellsgenetically modified to contain an expressible transgene coding forCFTR. The cells may be epithelial progenitor cells. The cells may be foruse in the treatment of cystic fibrosis.

The invention further teaches a process of preparing transformants ofmammalian cells, which comprises transfecting said mammalian cells withat least one gene coding for a factor selected from the group consistingof CFTR, PGIS, Ang-1, vascular endothelial growth factor family (VEGF A,B, C, PIGF), fibroblast growth factor, erythropoietin, hemoxygenase-1(HO-1) or hemoxygenase-2 (HO-2), transforming growth factor beta (orother member of the TGF-beta super family including BMPs 1, 2, 4, 7 andtheir receptors BMPR2 or BMPR1) and platelet derived growth factors (Aor B), to produce transformed cells capable of expressing said factor invivo. The mammalian cells may be selected from the group consisting ofendothelial cells, smooth muscle cells, progenitor cells such asendothelial progenitor cells (e.g. from bone marrow or peripheralblood), dermal fibroblasts, stem cells, mesenchymal stem cells, marrowstromal cells (MSC), epithelial cells, epithelial progenitor cells, andothers.

The invention further teaches a process of alleviating or inhibiting adisorder in a mammalian patient by conducting therapy which comprisesadministration to the lung by injection into the blood system of themammalian patient suffering from a disorder, of viable mammalian cells,wherein the mammalian cells are effective for tissue regeneration. Thedisorder may be a lung degenerative disorder. In embodiments of theinvention, the mammalian cells are selected from the group consisting ofprogenitor cells such as endothelial progenitor cells (e.g. from bonemarrow or peripheral blood), stem cells, mesenchymal stem cells, marrowstromal cells (MSC), epithelial cells and epithelial progenitor cells.The disorder may be pulmonary hypertension, chronic obstructivepulmonary disease, lung injury/ARDS, and pulmonary fibrosis.

In another embodiment, the invention teaches a process of alleviating orinhibiting pulmonary hypertension in a mammalian patient by conductingtherapy which comprises administration to the mammalian patient anangiogenic factor or a gene which expresses an angiogenic factor. Theangiogenic factor may be selected from the group consisting of vascularendothelial growth factor (VEGF) and its isoforms, fibroblast growthfactor (FGF, acid and basic), angiopoietin-1 and other angiopoietins,erythropoietin, hemoxygenase, transforming growth factor-α (TGF-α),transforming growth factor-β (TGF-β) or other members of the TGF-β superfamily including BMPs 1, 2, 4, 7 and their receptors MBPR2 or MBPR1,hepatic growth factor (scatter factor), and hypoxia inducible factor(HIF).

In one embodiment, the invention teaches a process of alleviating orinhibiting the progression of pulmonary hypertension in a mammalianpatient comprising administration to the lung by injection into thepulmonary circulation of the mammalian patient suffering from thedisorder, of fibroblast cells; said cells having been transformed invitro to express a transgene selected from the group consisting of:vascular endothelial growth factor, fibroblast growth factor,angiopoeitin, hemoxygenase, transforming growth factor, hepatic growthfactor, endothelial nitric oxide synthase, prostaglandin I synthase,Krupple-like factors (KLF-2, 4, and others) artificially engineeredtranscription factors providing the desired effects, and hypoxiainducible factor; and wherein said cells are further capable of lodgingin the small arteriolar-capillary transition regions of the circulatorysystem within the lungs, wherein the administration results inalleviation or inhibition of progression of the pulmonary hypertension.

In another embodiment, the invention teaches a process for alleviatingor inhibiting the progression of pulmonary hypertension in a mammalianpatient comprising administration to the lung by injection into thepulmonary circulation of the mammalian patient suffering from thedisorder, of smooth muscle cells, said cells having been transformed invitro to express a transgene selected from the group consisting of:vascular endothelial growth factor and endothelial nitric oxidesynthase; and wherein said cells are further capable of lodging in thesmall arteriolar-capillary transition regions of the circulatory systemwithin the lungs, wherein the administration results in alleviation orinhibition of progression of the pulmonary hypertension.

In yet another embodiment, the present invention teaches a process foralleviating or inhibiting the progression of pulmonary hypertension in amammalian patient comprising administration to the lung by injectioninto the pulmonary circulation of the mammalian patient suffering fromthe disorder, of cells selected from the group consisting of endothelialprogenitor cells and endothelial like progenitor cells. In oneembodiment, the cells may be allogenic. In another embodiment, the cellsmay be syngeneic. In another embodiment, the cells may be autologous. Ina further embodiment, the cells may be transformed in vitro to express atransgene, for example, an endothelial nitric oxide synthase. Theendothelial nitric oxide synthase may be human endothelial nitric oxidesynthase. The transformation may be through any known means; in oneembodiment, the transformation is through electroporation with eNOScloned into a plasmid vector. In one embodiment, the pulmonaryhypertension is associated with scleroderma. In another embodiment, thepulmonary hypertension is associated with congenital heart disease. Inanother embodiment, the pulmonary hypertension is associated with lupus(SLE). In another embodiment, the pulmonary hypertension is associatedor caused by idiopathic PAH.

In yet another embodiment, the present invention teaches a process foralleviating or inhibiting the progression of Acute Respiratory DistressSyndrome (ARDS) in a mammalian patient comprising administration to thelung by injection into the pulmonary circulation of the mammalianpatient suffering from the disorder, of mesenchymal stem cells. In oneembodiment, the cells are allogenic. In another embodiment, the cellsare autologous. In a further embodiment, the cells have been transformedin vitro to express a transgene. In one embodiment, the transgene isangiopoietin-1. The transformation may be through any known means; inone embodiment, the transformation is through electroporation withangiopoietin-1 cloned into a plasmid vector.

In another embodiment, the present invention teaches a process ofalleviating or inhibiting the progression of an acute respiratorydistress syndrome (ARDS) in a mammalian patient comprising administeringto the lung by injection into the pulmonary circulation of the patientof cells selected from the group consisting of smooth muscle cells,mesenchymal stem cells and fibroblast cells, said cells having beentransformed in vitro to express Angiopoietin-1, wherein theadministration results in alleviation or inhibition of the progressionof the ARDS. In one embodiment, the fibroblast cells are skinfibroblasts. In one embodiment, the cells are allogenic. In oneembodiment, the cells are syngenic. In one embodiment, the cells areautologous. The transformation may be through any known means; in oneembodiment, the transformation is through electroporation withangiopoietin-1 cloned into a plasmid vector.

In a further embodiment, the invention teaches a process of alleviatinga state selected from the group consisting of lung inflammation, septaledema, alveolar inflammation, and endothelial inflammation, in amammalian patient, comprising administering to the lung by injectioninto the pulmonary circulation of the patient of cells selected from thegroup consisting of smooth muscle cells, mesenchymal stem cells, andfibroblast cells, said cells having been transformed in vitro to expressAngiopoietin-1, wherein the administration results in alleviation of thestate. The state may be, for example, the result of or caused by acutelung injury. For example, the state may be associated with acuterespiratory distress syndrome.

In a further embodiment, the invention teaches a process of alleviatingor inhibiting the progression of pulmonary hypertension in a mammalianpatient comprising administration to the lung of a transgene selectedfrom the group consisting of: vascular endothelial growth factor,fibroblast growth factor, angiopoeitin, hemoxygenase, transforminggrowth factor, hepatic growth factor, endothelial nitric oxide synthase,Angiopoietin-1, prostaglandin I synthase (PGIS) and hypoxia induciblefactor; wherein the administration results in alleviation or inhibitionof progression of the pulmonary hypertension. In one embodiment, thetransgene is endothelial nitric oxide synthase. In another embodiment,the transgene is vascular endothelial growth factor. In anotherembodiment, the transgene is PGIS. In another embodiment, the transgeneis Angiopoietin-1. In another embodiment, the pulmonary hypertension isassociated with or caused by scleroderma, congenital heart disease,lupus (SLE), or idiopathic PAH.

In a further embodiment, the invention teaches a process for alleviatingor inhibiting the progression of Acute Respiratory Distress Syndrome(ARDS) in a mammalian patient comprising administration to the lung of atransgene selected from the group consisting of Angiopoietin-1 andvascular endothelial growth factor.

In a further embodiment, the invention teaches a process of alleviatinga state selected from the group consisting of lung inflammation, septaledema, alveolar inflammation, and endothelial inflammation, in amammalian patient, comprising administering to the lung a transgeneselected from the group consisting of Angiopoietin-1 and vascularendothelial growth factor. In one aspect, the state is a result of, orcaused by, acute lung injury. In another aspect, the state is associatedwith acute respiratory distress syndrome.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

A wide variety of transgenes encoding therapeutic factors can be used inthe processes and products of the present invention. While treatment ofpulmonary system disorders is a primary focus of the invention, it isnot limited to such treatments. Therapeutic factors expressed in thelung by the transgenes released into and delivered by the circulation ofother body organs downstream of the lungs are within the scope of thisinvention. Transgenes expressing therapeutic factors such as Factor VIIIfor treatment of classical haemophelia, and other clotting factors fortreating various bleeding disorders may be used. Other examples include:

transgenes expressing hormones, for example growth hormone for treatmentof hypopituitary dysfunction, insulin, (thyroid stimulating hormone(TSH) for treatment hypothyroidism following pituitary failure, andother hormones;

transgenes expressing beneficial lipoproteins such as Apo A1 and otherproteins/enzymes participating in lipid metabolism such as lipoproteinlipase;

transgenes expressing prostacyclin synthase or other transgenes thatproduce vasoactive substances;

transgenes expressing anti-oxidants and free radical scavengers;

transgenes expressing soluble cytokine receptors to neutralize actionsof damaging levels of immune mediators, for example soluble TNF∀receptor, or cytokine receptor antagonists, for example IL1ra;

transgenes expressing soluble adhesion molecules, for example ICAM-1, tointerrupt pathological cell adhesion processes such as those which occurin inflammatory diseases;

transgenes expressing soluble receptors for viruses to inhibit infectionof cells, e.g. CD4, CXCR4, CCR5 for HIV;

transgenes expressing cytokines, for example IL-2, to activate immuneresponses for combatting infections;

the cystic fibrosis gene, as a transgene.

Other examples of transgenes for use in the cell based therapy of theinvention include transgenes encoding for:

-   -   elastase inhibitors for use in treating pulmonary vascular        disease such as pulmonary hypertension or systemic vascular        disease;    -   tissue inhibiting metaloproteins for use in treating        atherosclerosis or arterial aneurysms    -   potassium channels or potassium channel modulators for use in        treating pulmonary hypertension    -   anti-oxidants such as superoxide dismutase for use in treating        pulmonary hypertension, ARDS and pulmonary fibrosis    -   anti-inflammatory factors such as cytokines, IL-10 and IL-4 for        use in treating inflammatory vascular disease such as        atherosclerosis or arterial aneurysms

The transfected cells lodged in the lung and containing transgenesexpressing such factors and other products will act as a systemic sourceof the appropriate factor.

In some instances, certain cell types, on their own, for exampleEndothelial Progenitor Cells, Mesenchymal Stem Cells, or EndothelialProgenitor-like Cells, can be therapeutic absent genetic modification tooverexpress, or express, these transgenes.

One preferred aspect of the present invention is the treatment ofpulmonary hypertension (PH). Primary pulmonary hypertension (PPH) andother causes of PH are associated with severe abnormalities inendothelial function, which likely play a critical role in itspathogenesis. The vasodilatory, anti-thrombotic and anti-proliferativefactor, nitric oxide (NO) has been demonstrated to decrease pulmonarypressures in both experimental and clinical situations. However,long-term viral-based methods may cause significant local inflammation.Other, previous attempts to treat PPH have involved the use ofprostacyclin, using continuous administration, but this is a difficultand expensive procedure, liable to give rise to side effects.

The present invention provides, from this second preferred aspect, amethod of alleviating the symptoms of PPH (and other causes of PH) whichcomprises administering to the pulmonary system of a patient sufferingtherefrom transformed mammalian fibroblast cells from dermal or otherorigins, endothelial cells or progenitor cells, i.e. EndothelialProgenitor Cells, or Endothelial Progenitor-like Cells derived from bonemarrow or isolated from the systemic circulation, said transfected cellsincluding at least one expressible transgene coding for an angiogenicfactor for release thereof into the pulmonary circulation.

Specific examples of useful angiogenic factors for delivery by way oftransgenes in cells, or by way of other routes of the additional aspectof this invention include vascular endothelial growth factor (VEGF) inall of its various known forms, i.e. VEGF165 which is the commonest andis preferred for use herein, VEGF205, VEGF189, VEGF121, VEGFB and VEGFC(collectively referred to herein as VEGF); fibroblast growth factor(FGF, acid and basic), angiopoietin-1 and other angiopoietins,transforming growth factor-α (TGF-α), transforming growth factor-β(TGF-β) or other members of the TGF-β super family, including BMPs 1, 2,4, 7 and their receptors BMPR2 or BMPR1, and hepatic growth factor(scatter factor) and hypoxia inducible factor (HIF). VEGF is thepreferred angiogenic factor, on account of the greater experience withthis factor and its level of effective expression in practice. Specificexamples of useful vasoactive factors for delivery by way of transgenesin cells, or by way of other routes of the additional aspect of thisinvention include nitric oxide synthase (NOS), endothelial nitric oxidesynthase (eNOS), PGIS, and hemoxygenase. DNA sequences constituting thegenes for these factors are known, and they can be prepared by thestandard methods of recombinant DNA technologies (for example enzymaticcleavage and recombination of DNA), and introduced into mammalian cells,in expressible form, by standard genetic engineering techniques such asthose mentioned above (viral transfection, electroporation, lipofection,use of polycationic proteins, etc).

In an additional aspect of the invention, angiogenic factors can beadministered directly to the patient, e.g. by direct infusion of thefactor, into the vasculature. They can also be administered to thepatient by processes of inhalation, whereby a replication-deficientrecombinant virus coding for the angiogenic factor is introduced intothe patient by inhalation in aerosol form, or by intravenous or arterialinjection of the DNA constituting the gene for the factor itself(although this is inefficient). Such administration methods, includinginjection or inhalation, can also be used for cells transfected with theangiogenic factors. Administration methods as used in known treatmentsof cystic fibrosis can be adopted.

Angiogenic factors such as those mentioned above have previously beenproposed for use as therapeutic substances in treatment of vasculardisease. It is not to be predicted from this work, however, that suchangiogenic factors would also be useful in treatment of pulmonaryhypertension. Whilst it is not intended that the scope of the presentinvention should be limited to any particular theory or mode ofoperation, it appears that angiogenic growth factors may also haveproperties in addition to their ability to induce new blood vesselformation. These other properties apparently include the ability toincrease nitric oxide production and activity, and/or decrease theproduction of endothelin-1, in the pulmonary circulation, so as toimprove the balance of pulmonary cell nitric oxide in endothelin-1production.

In preparing cells for transfection and subsequent introduction into apatient's pulmonary system, it is preferred to start with somaticmammalian cells obtained from the eventual recipient (i.e. autologouscells) of the cell-based gene transfer treatment of then presentinvention, however, it is also possible that in other instances theremay be advantages to using cells derived from another individual (i.e.allogenic cells). A wide variety of different cell types may be used,including fibroblasts, endothelial cells, smooth muscle cells,progenitor cells (e.g. from bone marrow or peripheral blood),fibroblasts, such as dermal fibroblasts, endothelial progenitor cells(EPCs), endothelial-like progenitor cells (ELPC's), mesenchymal cells,marrow stromal cells (MSC), and epithelial cells, and others. Dermalfibroblasts are simply and readily obtained from the patient's exteriorskin layers, readied for in vitro culturing by standard techniques.Endothelial cells are harvested from the eventual recipient, e.g. byremoval of a saphenous vein and culture of the endothelial cells.Progenitor cells can be obtained from bone marrow biopsies oraspiration, or isolated from the circulating blood, and cultured invitro. The culture methods are standard culture techniques with specialprecautions for culturing of human cells with the intent ofre-implantation. With certain cell types, such as MSCs, EPCs or ELPCs,the cells alone have efficacious properties without transfection, andmay be used either alone, or, for synergistic effect, transfected orotherwise expressing an angiogenic factor as described above. The EPCsMSCs or ELPCs may be autologous, syngenic, or even allogenic.

One embodiment of the present invention uses dermal fibroblasts from thepatient as the cells for gene transfer. Given the fact that the logicalchoice of cell types for one skilled in the art to make would be a celltype naturally found in the patient's pulmonary system, such as smoothmuscle cells, the use of fibroblasts is counter-intuitive. Surprisingly,it has been found that fibroblasts are eminently suitable for this work,exhibiting significant and unexpected advantages over cells such assmooth muscle cells. They turn out to be easier to grow in culture, andeasier to transfect with a transgene, given the appropriate selection oftechnique. They yield a higher proportion of transfectants, and a higherdegree of expression of the angiogenic factors in vivo, afterintroduction into the patient's pulmonary system. The anticipatedgreater risk with fibroblasts of possibly causing fibrosis in thepulmonary system, as compared with smooth muscle cells, has notmaterialized.

The somatic gene transfer in vitro to the recipient cells, i.e. thegenetic engineering, is performed by standard and commercially availableapproaches to achieve gene transfer, as outlined above. Preferably, themethod includes the use of poly cationic proteins (e.g. SUPERFECT™) orlipofection (e.g. by use of GENEFECTOR™), agents available commerciallyand which enhance gene transfer. However, other methods besideslipofection and polycationic protein use, such as, electroporation,viral methods of gene transfer including adeno and retro viruses, may beemployed. These methods and techniques are well known to those skilledin the art, and are readily adapted for use in the process of thepresent invention. Lipofection is a commonly used technique, for usewith dermal fibroblast host cells, whereas the use of polycationicproteins is preferred for use with smooth muscle cells. Electroporationcan also be used as it is more easily applied in the context of humantherapy. Different methods can be selected based on whether transent ormore permanent expression of the transgene is desired.

The re-introduction of the genetically engineered cells into thepulmonary circulation can be accomplished by infusion of the cellseither into a peripheral vein or a central vein, from where they movewith the circulation to the pulmonary system as previously described,and become lodged in the smallest arterioles of the vascular bed of thelungs. Direct injection into the pulmonary circulation can also beadopted, for example through a Swan Ganz catheter. Injection into theright ventricle or right atrium may be carried out using the pacing portof a Swan Ganz catheter. The infusion can be done either in a bolus formi.e. injection of all the cells during a short period of time, or it maybe accomplished by a continuous infusion of small numbers of cells overa long period of time, or alternatively by administration of limitedsize boluses on several occasions over a period of time. Re-introductionof genetically engineered cells into the lungs can also be accomplishedthrough inhalation of the cells using known pulmonary administrationmethods, such as an inhaler.

While the transfected cells themselves are largely or completelyretained in the pulmonary circulation, and especially in the arteriolesof the patient's lungs, the expression products of the transgenesthereof are not restricted in this manner. They can be expressed andsecreted from the transfected cells, and travel through the normalcirculation of the patient to other, downstream body organs where theycan exert a therapeutic effect. Thus, while a preferred use of theprocess of the invention is in the treatment of pulmonary disorders,since the expression products initially contact the patient's pulmonarysystem, it is not limited to such treatments. The vectors can containtransgenes expressing products designed for treatment of other bodyorgans of the patient. Such products expressed in the pulmonary systemwill target the other, predetermined organs and be delivered thereto bythe natural circulation system of the patient.

Another preferred embodiment is the treatment of Acute RespiratoryDistress Syndrome (ARDS). This ARDS results as a consequence of acutelung injury (ALI) that may have been caused by pneumonia, sepsis, oracute lung injury from another source. The treatment of the ARDS mayinclude decreasing lung inflammation, decreasing septal edema,decreasing alveolar and/or endothelial inflammation, or alleviatinganother symptom of the ARDS. With certain cell types, such asmesenchymal and stem cells, the administration of the cells alone maytreat the ARDS. Another embodiment comprises use of smooth muscle cellsMSCs or fibroblast cells such as dermal fibroblasts, transformed toexpress a transgene, for example, Ang-1.

The invention is further described for illustrative purposes, in thefollowing specific, non-limiting Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described, by way of example only, withreference to the accompanying drawings, in which:

FIG. 1: 1A illustrates fluorescence of pulmonary artery smooth musclecells immediately following incubation with the viable fluorophoreCMTMR, as described below in Example 2;

FIGS. 1B and 1C respectively illustrate multiple cell-shaped fluorescentsignals at fifteen minutes and 48 hours after jugular injection asdescribed in Example 5;

FIG. 2: 2A shows that a transfection efficiency of about 15% could beobtained with the primary pulmonary artery smooth muscle cells in vitro,discussed in Example 6;

FIGS. 2B and 2C respectively show the staining in the lung at 48 hoursand 14 days following injection, as described in Example 6;

FIG. 3 provides a graphic representation of right ventricular systolicpressure four weeks after monocrotaline injection and cell-based genetransfer as described in Example 7;

FIG. 4 provides a graphic representation of right ventricular to leftventricular plus septal weight ratio four weeks after monocrotalineinjection and cell-based gene transfer as described in Example 7;

FIG. 5: 5A illustrates the smooth muscle hypertrophic and hyperplasticresponse observed in mid-sized pulmonary vessels four weeks followingsubcutaneous injection of monocrotaline as described in Example 7;

FIG. 5B shows similar results as FIG. 5A in animals transfected with thecontrol vector, pcDNA 3.1 as described in Example 7;

FIG. 5C shows similar results as FIG. 5A following cell-based genetransfer of VEGF as described in Example 7;

FIG. 6 is a graphic representation of medial area followingmonocrotaline injection and gene transfer as described in Example 7;

FIG. 7 graphically represents results obtained by selectively amplifyingthe exogenous VEGF transcript as described in Example 7;

FIG. 8 provides a graphic representation of right ventricular systolicpressure following monocrotaline injection and delayed gene transfer asdescribed in Example 8; and

FIG. 9 provides a graphic representation of right ventricular to leftventricular plus septal weight ratio following monocrotaline injectionand delayed gene transfer (reversal experiments) as described in Example8.

FIG. 10 is a gel showing a band of 1.5 kb (arrowhead: lanes 1 and 2).

FIG. 11 is a bar graph showing cell-based gene transfer using PGIS andeNOS in experimental pulmonary hypertension.

FIG. 12 is a bar graph showing cell-based gene transfer using PGIS andeNOS in experimental pulmonary hypertension.

FIG. 13 is a bar graph showing cell-based gene transfer using VEGF oreNOS in experimental pulmonary hypertension.

FIG. 14 is a gel showing the results of multiple transfections using thecDNA for eNOS.

FIG. 15 are photographs comparing a single transfection to a doubleprotocol.

FIG. 16 is a photograph which indicates the morphology of isolated lungepithelial cells in primary cell culture, 5 days after isolation.

FIG. 17 is a photograph which shows fluorescent microscopy showingpurity of isolated lung epithelial cells.

FIG. 18 is a bar graph showing decrease in Wet/Dry lung weight by use ofgene therapy.

FIG. 19 is a bar graph showing decrease in peak airway pressure by useof gene therapy.

FIG. 20 is a bar graph showing maintenance of partial oxygen pressure ascompared to the null vector, by use of gene therapy.

FIG. 21 is a bar graph showing that dosing cell-based endothelial NOSgene transfer inhibits MCT-induced PH and the effect of multipleinjections, measured by RVSP.

FIG. 22 is a bar graph showing that dosing cell-based endothelial NOSgene transfer inhibits MCT-induced PH and the effect of multipleinjections, measured by RV/LV+S.

FIG. 23 is a bar graph showing that dosing cell-based endothelial NOSgene transfer inhibits MCT-induced PH and the effect of multipleinjections, measured by weight gain.

FIG. 24 (a-d) are a series of photographs of ELPC phenotype in vitro.FIG. 24 (e-g) show sections of lung 3 days post-administration of ELPCs:e shows ELPC cells trapped within distal arterioles; f shows engraftingof ELPCs into the endothelial layer of distal precapillary arterioles. gshows complete luminal incorporation. FIG. 24 (h-i) show sections oflung 21 days post-administration.

FIG. 25 is a bar graph showing the effect of early ELPC injection onright ventricular systolic pressure (A) and the ratio of rightventricular to left ventricular weight (RV/LV)(B) at end study (day21)(prevention protocol). Rats treated with ELPCs exhibited asignificantly lower RVSP with no significant decrease in RV/LV ratio(P<0.001).

FIG. 26 shows persistence of effect over a longer time period. RSVP inrats injected with fibroblasts versus ELPCs are shown at day 21 (A) andat a longer time periods (B). FIG. 26C shows histological examination ofkidneys (a, b, c, d) and lungs (e-f) in MCT-ELPC-treated rats (b, d, e)versus controls (a, c, f).

FIG. 27(A) shows RVSP at 21 days after MCT and 14 days after celltherapy (day 35), in animals treated with MCT, ELPC, or ELPC cellstransduced with and expressing eNOS. ELPC inhibited progression of RSVP,with eNOS-transduced ELPC's resulting in significantly lower RSVPlevels. Rats receiving eNOS-ELPCs or ELPCs alone had markedly reducedhypertrophy (B).

FIG. 28A shows representative confocal projection images of lungsections perfused with fluorescent microspheres suspended in agarose andimmunostained for α—smooth muscle action (SMA). a: control rats; b: ratstreated with MCT (21 days); c: animals receiving ELPCs (preventionmodel); d: rats treated with MCT (35 days); e: animals receiving ELPCs(reversal model); f: animals receiving eNOS-transduced ELPCs.

FIG. 28B shows summary data for pulmonary microvascular perfusion foranimals treated in the prevention (open bars) and reversal (closed bars)protocols (LEPC treated and eNOS-transduced ELPC treated animals).

FIG. 28C shows proportion of small pulmonary arterioles that arenonmuscularized (NM), partially muscularized (PM) or fully muscularized(FM) in the reverse protocol (ELPC treated and eNOS-transduced ELPCtreated animals).

FIG. 29 shows survival to 35 days in a reversal study (animals treatedwith MCT, ELPC cells, or eNOS-transduced ELPC cells).

FIG. 30 shows the therapeutic potential of MSCs, alone or transfectedwith pAng1, on LPS induced lung inflammation in mice.

FIG. 30A shows the total cell count on BAL fluid to evaluate lungairspace inflammation. There was a 19-fold increase in totalinflammatory cells in BAL fluid collected 3 days after LPS, which wasreduced by 53% in MSCs-treated mice (non-/null-transfected), and by 96%with Ang-1-transfected MSCs (MSCs-pAngI). Group comparisons wereanalyzed by one-way ANOVA with Dunnett's post hoc test. * p<0.05 and **p<0.01, compare between LPS/Saline vs. each treated group (MSC5,MSC5-pFLAG, or MSCs-pAngI). n=5 per group.

FIG. 30B shows histological evaluation of therapeutic potential of MSCsand MSC-pAng1 on LPS-induced lung injury in mice. Representative imagesof hematoxylin and eosin stained lung sections from six experimentalgroups. Lungs were inflation-fixed with 4% paraformaldehyde, paraffinembedded, and then cut into 5-micron thick sections before beingstained. Photomicrographs were obtained with a Nikon Eclipse E800microscope with a 40× objective. Scale bar 20 μm.

FIG. 31 shows the levels of pro-inflammatory cytokines and chemokines inBAL fluid. Levels of the pro-inflammatory cytokines, IFN-γ, TNF-α, IL-6and IL-1β, in BAL fluid, were measured using ELISA. Chemokine levels(MIP-2, JE [murine MCP-1 homologue], and KC [murine IL-8 homologue]), inBAL fluid were measured by multiplex immunoassay. Group comparisons wereanalyzed by one-way ANOVA with Dunnett's post hoc test. * p<0.0 and **p<0.01, LPS/Saline vs. each treated group (MSCs, MSCs-pFLAG, orMSCs-pAng1). n=5 per group.

FIG. 32 shows levels of pro-inflammatory cytokines and chemokines inlung homogenate. Cytokine (TNF-a and IL-6) and chemokine (MIP-2, JE[murine MCP-1 homologue], and KC [murine IL-8 homologue]) levels in lunghomogenates were measured by multiplex immunoassay. Group comparisonswere analyzed by one-way ANOVA with Dunnett's post hoc test. * p<0.05and ** p<0.01, LPS/Saline vs. each treated group (MSCs, MSCs-pFLAG, orMSCs-pAng1). n=5 per group.

FIG. 33 shows the effect of MSCs and MSCs-pAng1 on LPS-induced ALI.Therapeutic efficacy was assessed by measurement of total protein,albumin, and 1 gM (biomarkers of pulmonary vascular leak resulting fromdisruption of the alveolar-capillary membrane barrier) in BAL fluid.FIG. 33A shows total protein concentration was measured by Bradfordassay; FIG. 33B shows albumin, measured using a mouse-specific albuminELISA; and FIG. 33C shows IgM as measured using a mouse IgM ELISA kit.Group comparisons were analyzed by one-way ANOVA with Dunnett's post hoctest. * p<0.05 and ** p<0.01, LPS/Saline vs. each treated group (MSCs,MSCs-pFLAG, or MSCs-pAng1). n=5 per group.

FIG. 34 shows retention of injected MSCs in mice with or withoutLPS-induced ALI. MSCs were labeled with the cell tracing dye CFDA SE(green) prior to injection. Nuclei were stained with TO-PRO-3 (blue).FIG. 34A shows labelled MSCs (indicated by white arrows) in 5-μm,PFA-fixed lung sections from LPS-injured mice sacrificed at 15 minutes(initial retention). FIG. 34B shows three-dimensional lung section froman animal that received labelled MSCs. Lung was inflated, stored in OCT,and cut into 50-μm thick section. Z-series images (30 sections, totalthickness of the tissue scanned=17.71 μm) were collected with a 63× oilobjective and projected in different axes, as shown. FIG. 34C showspictures taken in z axis with a 20× objective, then stacked using LeicaConfocal software. FIG. 34D shows flow cytometry of lung lobes (leftupper and all right lobes), enzyme-digested into single cells thenanalyzed. n=5 per group. Scale bar=20 μm.

FIG. 35 shows cell-based gene transfer restores Angiopoietin-1expression in a rat model of ALI. A, panels a and b showimmunofluorescent staining for von Willebrand Factor and CMTMR-labeledAng-1 transfected fibroblasts. A, panels c and d show immunofluorescentstaining for Ang-1. A proportion of CMTMR-labeled Ang-1 transfectedfibroblasts cells also expressed Ang-1 48 hours after injection (d).FIG. 1B shows quantitative real-time RT-PCR analysis of total Ang-1 mRNAlevels, and shows a 53% reduction following LPS exposure that wasrestored by pretreatment with pAng-1 transfected cells. FIG. 1C showsthat plasmid Ang-1 mRNA was undetectable in animals pretreated withpFLAG-transfected cells, while plasmid Ang-1 levels were similar in bothgroups that receive injection of pAng-1-transfected cells. FIG. 1D showsquantitative real-time RT-PCR analysis of Tie2 mRNA levels. The figuresshow a 59% reduction following LPS exposure that was partially restoredby pretreatment with pAng-1 transfected cells. FIG. 1E showsimmunoprecipitation and Western Blot analysis demonstrating that bothTie2 protein and phosphorylated Tie2 protein were decreased followingLPS exposure and partially restored by pretreatment with pAng-1transfected cells. * denotes significance of differences vs.pFLAG-transfected fibroblast injected rats challenged with saline;p<0.05. # denotes significance of differences vs. pFLAG-transfectedfibroblast injected rats challenged with LPS; P<0.05. N=10/group. Scalebars=100 μm.

FIG. 36 shows Angiopoietin-1 cell therapy attenuating intra-alveolarseptal thickness and airspace inflammation in rats. FIG. 2A showsrepresentative hematoxylin and eosin stained lung sections demonstratingnormal lung morphology in saline challenged rats after injection ofeither pFLAG-transfected cells (panel a) or pAng-1-transfected cells(panel b). The increased edema and infiltration observed in LPSchallenged rats pretreated with pFLAG-transfected cells (panel c) wasreduced in LPS challenged rats pretreated with pAng-1-transfected cells(panel d). FIG. 2B shows quantification of intra-alveolar septalthickness demonstrating that the LPS-induced 2-fold increase in septalthickness was significantly attenuated by pretreatment withpAng-1-transfected cells. FIG. 2C shows that the total number of cellsin BALF was increased 4-fold following LPS challenge and pretreatmentwith pFLAG-transfected cells, but was significantly reduced bypretreatment with pAng-1-transfected cells. FIG. 2D shows that totalprotein in BALF was increased 73% following challenge and pretreatmentwith pFLAG-transfected cells and tended to be reduced by pretreatmentwith pAng-1-transfected cells. FIG. 2E shows that lung wet weight tobody weight ration was increased 20% following LPS challenge andpretreatment with pFLAG-transfected cells and tended to be reduced bypretreatment with pAng-1-transfected cells. * denotes significance ofdifferences vs. pFLAG-transfected fibroblast injected rats challengedwith saline; p<0.05. # denotes significance of differences vs.pFLAG-transfected fibroblast injected rats challenged with LPS; p<0.05.N=10/group. Scale bar=100 μm.

FIG. 37 shows the effect of angiopoietin-1 cell therapy on endothelialadhesion molecule expression in rats. FIG. 3A shows quantitativereal-time RT-PCR analysis of total ICAM-1 mRNA levels, which showed a2-fold increase following LPS that was not affected by pretreatment withpAng-1-transfected cells. Western blot analysis of ICAM-1 showed nodetectable difference in ICAM-1 expression between all experimentalgroups. FIG. 3B shows quantitative real-time RE-PCR analysis of totalVCAM-1 mRNA levels showing a 2-fold increase following PLS that was notaffected by pretreatment with pAng-1-transfected cells. Western blotanalysis of VCAM-1 showed no detectable difference in VACM-1 expressionbetween all experimental groups. FIG. 3C shows quantitative real-timeRT-PCR analysis of total E-Selectin mRNA levels showing a 22-foldincrease following LPS that was significantly attenuated by pretreatmentwith pAng-1-transfected cells. Western blot analysis of E-Selectinshowed similar expression to mRNA levels. FIG. 3D shows quantitativereal-time RT-PCR analysis of total P-Selectin mRNA levels showing a32-fold increase following LPS that was not affected by pretreatmentwith pAng-1-transfected cells. Western blot analysis of P-Selectin wasnot performed due to lack of commercially-available antibodies. *denotes significance of differences vs. pFLAG-transfected fibroblastinjected rats challenged with saline; p<0.05. # denotes significance ofdifferences vs. pFLAG-transfected fibroblast injected rats challengedwith LPS: p<0.05. N=10/group.

FIG. 38 shows angiopoietin-1 and Tie2 expression in a transgenic mousemodel of ALI. FIG. 4A shows Western Blot analysis demonstrating thatAng-1 was reduced wildtype mice following LPS challenge compared tonaïve but restored to naïve levels in both Ang-1-tTA binary transgenicand Tie2+/− mice. FIG. 4B shows that Tie2 protein is lower in Tie2haploinsufficient mice compared to wildtype and Ang-1-tTA mice. Tie2protein was reduced in wildtype mice and Ang-1-tTA mice following LPSchallenge compared to naïve, but remained higher compared to Tie2+/−.

FIG. 39 shows septal thickness and airspace inflammation in transgenicmice. FIG. 5A shows representative hematoxylin and eosin stained lungsections, demonstrating normal lung morphology in naïve wildtype mice(panel a), Ang-1-tTA binary transgenic mice (panel b) and Tie2+/− mice(panel c). The increased edema and infiltration observed in LPSchallenged wildtype mice (panel d) was attenuated in Ang-1-tTA binarytransgenic mice (panel e) and exacerbated in Tie+/− mice (panel f). FIG.5B shows quantification of intra-alveolar septal thickness demonstratingthat the LPS-induced 2.7-fold increase in septal thickness issignificantly attenuated in Ang-1-tTA binary transgenic mice andexacerbated in Tie2+/− mice. FIG. 5C shows the total number of cells inBALF, which was increased 11-fold following LPS challenge in wildtypemice, but was significantly reduced in Ang-1-tTA mice and increased inTie2+/− mice. FIG. 5D shows that total protein in BALF was increased3-fold following LPS challenge in wildtype mice and was significantlyreduced in Ang-1-tTA mice and increased in Tie2+/− mice. * denotessignificance vs. wildtype naïve mice; p<0.05. # denotes significance ofdifferences vs. wildtype LPS challenged mice; p<0.05. N=5/group. Scalebar=100 μm.

FIG. 40 shows proinflammatory cytokines in BALF of transgenic mice. FIG.6A shows that the LPS-induced increase TNF-α was significantlyattenuated in Ang-1-tTA binary transgenic mice compared to wildtype.FIG. 6B shows that the increased LPS-induced increase in BALF IL-β wassignificantly attenuated in Ang-1-tTA mice compared to wildtype. FIG. 6Cshows that the LPS-induced increase in BALF IL-6 was significantlyattenuated in Ang-1-tTA binary transgenic mice and significantlyexaggerated in Tie2+/− mice compared to wildtype. * denotes significanceof differences vs. wildtype naïve mice; p<0.05. # denotes significanceof differences vs. wildtype LPS challenged mice; p<0.05. N=5/group.

FIG. 41 shows that Ang-1 overexpression reduces endothelial expressionof adhesion molecules. FIG. 7A shows flow cytometric analysis, showingthat the percentage of endothelial cells positive for E-Selectinexpression increased 34-fold following LPS in wildtype mice and wasreduced in Ang-10-tTA mice (left panel). The majority of total lungcells expressing E-Selectin were endothelial cells (right panel;endothelial cells indicated by open bars; non-endothelial cellsindicated by closed bars). FIGS. 7B, C and D show that similarly,percentage of endothelial cells positive for P-Selectin, ICAM-1 andVCAM-1 expression was increased following LPS in wildtype mice and thiswas reduced in Ang-1-tTA transgenic mice (left panels). Again, themajority of total lung cells expressing adhesion molecules wasendothelial cells (right panels; endothelial cells indicated by openbars; non-endothelial cells indicated by closed bars). * denotessignificance of differences vs. wildtype naïve mice; p<0.05. # denotessignificance of differences vs. wildtype LPS challenged mice; p<0.05.N=5/group.

FIG. 42 shows that Tie2 deficiency increased circulating levels ofsoluble adhesion molecules. FIGS. 8A, B and C show that solubleE-Selectin, P-Selectin and ICAM-1 were increased following LPS inwildtype mice and these were reduced in Ang-1-tTA binary transgenic miceand exaggerated in Tie2+/− mice. FIG. 8D shows that soluble VCAM-1 wasunchanged following LPS in WT and Ang-1-tTA mice, but increased inTie2+/− mice exposed to LPS compared to all other groups. * denotessignificance of difference vs. wildtype naïve mice; p<0.05. # denotessignificantly different wildtype LPS challenged mice; p<0.05. N=5/group.

EXAMPLE 1 Pulmonary Artery Explant Culture

Fisher 344 rats (Charles River Co.) were obtained at 21 days of age andwere sacrificed by overdose with ketamine and xylazine. The mainpulmonary artery was excised and transferred immediately into aphosphate-buffered saline (PBS) solution containing 2% penicillamine andstreptomycin (Gibco BRL, Burlington, Ontario). The adventitia wascarefully removed with sterile forceps, the artery opened longitudinallyand the endothelium removed by abrasion of the intimal surface with ascalpel. The vessel was cut into approximately 4 millimeter squarepieces which were placed intimal surface down on individualfibronectin-coated (Sigma Chemical Co., Mississauga, Ontario) tissueculture plates (Falcon, Becton Dickinson Canada, Mississauga, Ontario).The explants were then grown in Dulbecco's Modified Eagle Media with 10%fetal calf serum (FCS) and 2% penicillamine and streptomycin (all GibcoBRL), in a humidified environment with 95% O2 and 5% CO2 at 37EC, withthe media being changed every second day. Explants were passaged using0.05% trypsin/EDTA (Gibco BRL) once many cells of a thin, fusiformsmooth muscle cell phenotype could be clearly seen growing from thepulmonary artery segment, at which time the remaining explanted tissuewas removed. The cells were then grown in DMEM with 10% FCS and 2%penicillamine and streptomycin until they were to be used in furtherexperiments.

EXAMPLE 2 Alpha-Smooth Muscle Actin and Von Willebrand FactorFluorescent Staining

To confirm their smooth muscle cell identity and rule out endothelialcell contamination, cells at the third passage were plated onto coverslips and grown until 70% confluent, at which time they were fixed inacetone at room temperature for 10 minutes. The cells were incubatedwith FCS for 30 minutes at 37° C. to block non-specific bonding sites,and then with a monoclonal anti-alpha-actin antibody (5micrograms/millilitre) (Boehringer Mannheim) and a rabbit-derivedpolyclonal anti-von Willebrand Factor antibody (1:200 dilution) (Sigma)for 60 minutes at 37° C. in a covered humidified chamber. Negativecontrol cover slips were incubated with PBS for the same duration oftime. The cover slips were then washed in PBS, and incubated for 60minutes at room temperature in a PBS solution containing aCy3-conjugated donkey anti-mouse IgG antibody (1:200 dilution) (JacksonImmunoResearch Laboratories), a fluorescein isothiocyanate(FITC)-conjugated goat anti-rabbit IgG antibody (1:200) (JacksonImmunoResearch Laboratories), and Hoescht 33258 (Sigma), a fluorescentnuclear counterstain. The cover slips were again washed with PBS, andmounted using a 1:1 solution of PBS and gycerol. Slides were examinedusing an Olympus BX50 epifluorescent microscope with standardfluorescein, rhodamine and auto-fluorescent emission and excitationfilters. For each cover slip the immunofluorescence for action, vWF, andfor the nuclear counterstain Hoescht was indicated as positive ornegative.

All of the explant derived cultures were found to be at least 97% puresmooth muscle cell with very rare endothelial contamination. This couldbe attributed to the vigorous debridement of the endothelial liningduring the initiation of the explant, and early passaging with removalof the residual explant material.

Fluorescent Cell Labeling—Cells between the fifth and ninth passageswere grown until 80% confluent and were then labeled with the viablefluorophore, chloromethyl trimethyl rhodamine (CMTMR, Molecular ProbesInc., Eugene, Oreg.). CMTMR affords a very accurate method of detectingex vivi labeled cells, as the molecule undergoes irreversibleesterification and glucoronidation after passing into the cytoplasm of acell and thereby generates a membrane-impermeable final product. Thisactive fluorophore is then unable to diffuse from the original labeledcell into adjacent cells or structures, and may be detected in vivo forseveral months, according to the manufacturer. The fluorescent probe wasprepared by dissolving the lyophilized product in dimethyl sulfoxide(DMSO) to a concentration of 10 millimolar. This solution was stored at−20° C., an diluted to a final concentration of 25 micromolar inserum-free DMEM immediately prior to use. Cells were exposed to thelabeling agent for 45 minutes, and were then washed with PBS twice andthe regular media (DMEM with 10% FCS and 2% penicillin and streptomycin)replaced. The cells were grown overnight and harvested 24 hours laterfor injection into the internal jugular vein of recipient Fisher 344rats.

A series of in vitro experiments was also performed by plating the cellson cover slips and the incubating them with the fluorophore to determinethe quality and duration of fluorescence over time. Immediately afterincubation with the fluorophore, CMTMR, at a concentration of 25micromolar, 100% of cultured cells were found to fluoresce intenselywhen examined under a rhodamine filter (FIG. 1A). The white scale bar inFIG. 1A is 50 microns in length. Cells were also examined 48 hours and 7days after labeling, and despite numerous cell divisions 100% of thecells present on the cover slip continued to fluoresce brightly (datanot shown).

EXAMPLE 3 Ex Vivo Cell Transfection with the CMV-βGal Plasmid

The vector CMV-βGal (Clontech Inc., Palo Alto, Calif.), which containsthe beta-galactosidase gene under the control of the cytomegalovirusenhancer/promoter sequence, was used as a reporter gene to follow thecourse of in vivo transgene expression. The full-length coding sequenceof VEGF165 was generated by performing a reverse transcriptionpolymerase chain reaction using total RNA extracted from human aorticsmooth muscle cells and the following sequence specific primers: sense5′ TCGGGCCTCCGAAACCATGA 3′ (SEQ ID. NO. 1), antisense 5′CCTGGTGAGAGATCTGGTTC 3′ (SEQ ID. NO. 2), This generated a 649 bpfragment which was cloned into the pGEM-T vector (Promega, Madison,Wis.), and sequenced to confirm identity. The fragment was then clonedinto the expression vector pcDNA 3.1 at the EcoR1 restriction site, andcorrect orientation determined using a differential digest. The insertdeficient vector (pcDNA 3.1) was used as a control for the monocrotalineexperiments. All plasmid DNA was introduced into a JM109 strain of E.Coli via the heat-shock method of transformation, and bacteria wascultured overnight in LB media containing 100 micrograms/millilitre ofampicillin. The plasmid was then purified using an endotoxin-freepurification kit according to the manufacturer's instructions (QiagenEndotoxin-Free Maxi Kit, Qiagen Inc., Mississauga, Ontario), producingplasmid DNA with an A260/A280 ratio of greater than 1.75, and aconcentration of at least 1.0 micrograms/microliter. Smooth muscle cellsbetween the fifth and ninth passages were transfected using Superfect(Qiagen Inc., Mississauga, Ontario). This method was used to avoid theuse of viral vectors and simultaneously obtain significant in vitrotransfection efficiencies. The Superfect product is composed of chargedpolycations around which the plasmid DNA coils in a manner similar tohistone-genomic DNA interactions. This Superfect-DNA complex theninteracts with cell surface receptors and is actively transported intothe cytoplasm, after which the plasmid DNA can translocate to thenucleus. This technique allows the transfection reaction to be performedin the presence of serum (an important consideration in sensitiveprimary cell lines), and produces no toxic metabolites.

Cells between the fifth and ninth passages were trypsinized the dayprior to transfection to obtain a density of 5×10⁵ cells/dish. Thefollowing day, 5 micrograms of plasmid DNA was mixed with 300microlitres of serum-free DMEM in a sterile microcentrifuge tube. Theplasmid-media solution was then vortexed with 50 microlitres ofSuperfect transfection agent (Qiagen), after which the tubes wereincubated for 10 minutes at room temperature. The transfection mixturewas then combined with 3 milliliters of DMEM with 10% FCS and 2%penicillin and streptomycin and applied to the culture dishes after thecells had been washed with PBS. The solution was allowed to incubate at37° C. for 4 hours, and the cells were then washed with PBS twice andthe standard media replaced. The transfected cells were allowed to growovernight and were then harvested 24 hours later for animal injection.For every series of transfection reactions that were performed, one 100millimeter dish of pulmonary artery smooth muscle cells was stained invitro, to provide an estimate of the transfection efficiency of thetotal series.

In a total of 15 separate transfection reactions using the pCMV-α Galplasmid, an average transfection efficiency of 11.4% was obtained withthe primary pulmonary artery smooth muscle cells. No staining was seenin mock transfected cultures.

EXAMPLE 4 Animal Surgery

All animal procedures were approved by the Animal Care Committee of St.Michael's Hospital, Toronto, Canada. Six week old Fisher 344 rats(Charles River Co., St. Constant, Quebec) were anesthetized byintraperitoneal injection of xylazine (4.6 milligrams/kilogram) andketamine (70 milligrams/kilogram), and the cervical area shaved andcleaned with iodine and ethanol. A midcervical incision was made with ascalpel and the right internal, external and common jugular veinsidentified. Plastic tubing of 0.02 millimetres external diameter wasconnected to a 23 gauge needle and flushed with sterile saline (Baxter).Thus tubing was then used to cannulate the external jugular vein and wasintroduced approximately 5 centimetres into the vein to what wasestimated to be the superior vena caval level, and rapid venous bloodreturn was used to confirm the catheter location.

For experiments to determine the time course of cell survival andtransgene expression in the lung, pulmonary artery smooth muscle cellswhich had been labeled with the fluorophore CMTMR, or transfected withthe plasmid vector CMV-α Gal, were trypsinized, and centrifuged at 850rpm for 5 minutes. The excise media was removed and the pellet of cellswas resuspended in a total volume of 2 millilitres of phosphate-bufferedsaline (PBS). A 50 microlitre aliquot of these resuspended cells wasthen taken and counted on a hemocytometer grid to determine the totalnumber of cells present per millilitre of PBS. The solution was thendivided into 1 millilitre aliquots of approximately 500,000 cells andtransferred in a sterile manner to the animal care facility. These cellswere then resuspended by gentle vortexing and injected into the animalsvia the external jugular vein catheter. The solution was infused slowlyover one to two minutes and the catheter was then flushed again withsterile saline prior to removal. The external jugular vein was ligated,the incision closed with 3-0 interrupted absorbable sutures, and theanimals allowed to recover from surgery.

EXAMPLE 5 Detection of Fluorescently-Labeled Cells in Tissue

At 15 minutes, 48 hours, 7 days, or 14 days after delivery of labeledcells (n=5 for each time-point except for 15 minutes where n=4), orsaline injection (negative control, n=6), the animals were sacrificed byanesthetic overdose, and the chest cavity was opened. The pulmonaryartery and trachea were flushed with saline, and the right and leftlungs excised. Transverse slices were taken from the basal, medial andapical segments of both lungs, and specimens obtained from the liver,spleen, kidney and gastroenemius muscle. Tissue specimens were embeddedin OCT compound (Sakura Finetek U.S.A. Inc., Torrance, Calif.) en face,and then flash frozen in liquid nitrogen. Ten micron sections were cutfrom these frozen blocks at 2 different tissue levels separated by atleast 200 microns, and these sections were then examined under afluorescent microscope using a rhodamine filter, and the number ofintensely fluorescing cells was counted in each en face tissue specimen.

To provide an estimate of the total number of labeled cells presentwithin the entire lung, the total number of fluorescent cells werecounted in each lung section and averaged over the number of sectionscounted. A mathematical approximation could be made of the total numberof cells present within the lung by utilizing Simpson's rule for thevolume of a truncated cone. This equation bases the total volume of acone on the relative areas of 3 different sections such that:

volume=[(areabasal section+areamiddle section)×height of thelung]/3+[areaapical section/2×height of the lung/3]+[8/6×(height of thelung/3)3].

The height of the lung was measured after organ harvesting, and the areaof each transverse section was determined by planimetry. The averagenumber of cells present in the three sections, divided by the totalvolume of these sections yielded an estimate of the cell number per unitvolume. By multiplying this number by the total lung volume an estimateof the total number of cells within the lung could be obtained. Tocorrect for the appearance of a single cell in multiple adjacent lungsections, rats were injected with 500,000 CMTMR labeled cells andsacrificed acutely. The lungs were prepared, harvested and embedded inthe usual manner, and twenty serial sections, each 5 microns inthickness, were taken through the lung parenchyma. Each section wasexamined using a rhodamine filter and distinct individual cells wereidentified and their presence determined on adjacent sections. Thenumber of 5 micron sections in which a single cell could be identifiedwas counted and the average dimensions of a pulmonary artery smoothmuscle cell in vivo was obtained. The average diameter observed was16.4±1.22 microns. Therefore, the total number of cells calculated usingthe Simpson's formula was multiplied by 0.61 to correct for the presenceof 1 cell in, on average, each 1.64 ten micron sections.

Approximately 57±5% of the labeled cells could be identified within thelung 15 minutes after intravenous delivery, as shown by white arrows inFIG. 1B. Most of these cells appeared to be lodged in the capillarycirculation at the alveolar level. By 48 hours after cell delivery, asignificant decrease in the total number of fluorescent cells identifiedwas noted (34±7%, p<0.01), and the location of the cells also appearedto have changed. Many bright fluorescent signals were now identifiedwithin the pulmonary parenchyma, or were lodged within the wall of smallvascular structures as shown by the white arrows in FIG. 1C. The whitescale bar in FIGS. 1B and 1C is 50 microns in length. At 7 and 14 daysafter injection, a further decrease in cell number was noted (16±3% and15±5% respectively, both p<0.001 as compared to 15 minute time-point),however the cells appeared to remain in approximately the same location.No brightly fluorescent signals were seen in any of the lungs injectedwith non-labeled smooth muscle cells.

In the spleen, liver and skeletal muscle tissue no fluorescent signalswere identified. In 2 out of 4 kidneys examined at 48 hours followinginjection, irregular fluorescent signals could be identified. None ofthese appeared to conform to the shape of a whole cell, and werepresumed to represent those cells that were sheared or destroyed duringcell injection or shortly thereafter. In addition, no fluorescentsignals were identified in any organ outside of the lung 7 days afterinjection.

EXAMPLE 6 Detection of Beta-Galactosidase Expression in Tissue

At three time-points after cell-based gene transfer (48 hours, 7 days,and 14 days), animals (n=7 for each time-point) were sacrificed and thechest opened. The pulmonary artery was flushed with saline and thetrachea was cannulated and flushed with 2% paraformaldehyde until thelungs were well inflated. Transverse slices were taken from the basal,medial and apical segments of both lungs, and specimens obtained fromthe liver, spleen, kidney and gastroenemius muscle of certain animals.The specimens were incubated in 2% paraformaldehyde with 0.2%glutaraldehyde for 1 hour, and then rinsed in PBS. The tissue was thenincubated for 18 hours at 37° C. with a chromogen solution containing0.2% 5-bromo-4-chloro-3-indolyl-α-D-galactoside (X-Gal, BoehringerMannheim, Laval, Quebec), 5 millimolar potassium ferrocyanide (Sigma), 5millimolar potassium ferricyanide (Sigma), and 2 millimolar magnesiumchloride (Sigma), all dissolved in phosphate buffered saline. Thespecimens were then rinsed in PBS, embedded in OCT compound (MilesLaboratories), cut into 10 micron sections, and counterstained withneutral red.

The en face sections were examined microscopically, and the number ofintensely blue staining cells was determined. As one dish of cells wasused for in vitro staining to determine the transfection efficiency foreach reaction series, an estimate of the percentage of cells that weretransfected with the reporter gene plasmid pCMV-α Gal could be made forevery animal. Using this information and the mathematical calculationdescribed for approximating the number of fluorescent cells present, anestimate could be made of the total number of transfected cellsremaining at the time of animal sacrifice.

In a total of 15 separate transfection reactions using the pCMV-α Galplasmid, an average transfection efficiency of 13±0.5% was obtained withthe primary pulmonary artery smooth muscle cells in vitro, and is 15% inFIG. 2: 2A. No staining was seen in mock transfected cultures.

Following incubation with the X-Gal chromogen solution, microscopicevidence of cell-based transgene expression could be clearly seen at 48hours after injection of pCMV-α Gal transfected smooth muscle cells intothe internal jugular vein (n=7), with multiple intense blue stainingcells being seen throughout the lung (FIG. 2B), representingapproximately 36±6% of the original transfected cells that wereinjected. As with the fluorescently-labeled cells, most of thebeta-galactosidase expressing cells appeared to be lodged within thedistal microvasculature. For example, in FIG. 2B, the staining cells arepredominantly located in alveolar septae adjacent to small vessels,indicated by black arrows. By seven days after injection (n=4), adecline in the number of beta-galactosidase positive cells was noted(28±6%), and the intensity of staining also appeared to decrease. Again,the cells appeared to have either migrated into the pulmonary parenchymaor vascular wall. Fourteen days (n=6) after cell-based gene transfer, nofurther decrease in the number of cells identified was noted, but theintensity of beta-galactosidase staining of each cell had decreasedfurther, as shown by the black arrows in FIG. 2C, which shows theremaining cells apparently located within the pulmonary parenchyma. Theblack scale bar in FIGS. 2A to 2C is 50 microns in length. No evidenceof beta-galactosidase expression was detected in any of the lungs fromanimals (n=4, 3 at 7 days and 1 at 14 days) injected withnon-transfected smooth muscle cells. At all three time-points, noevidence of pulmonary pathology, as determined by the presence of anabnormal polymorphonuclear or lymphocytic infiltrate, septal thickeningor alveolar destruction, could be detected.

In the spleen and skeletal muscle of animals injected with transfectedor non-transfected smooth muscle cells, no blue staining cells could beidentified. Liver and renal specimens from animals injected with eithertransfected (n=5) or non-transfected (n=3) smooth muscle cells wouldoccasionally show faint blue staining across the cut edge of the tissue(n=2 for each group), but no intense staining was seen at anytime-point, and no staining was seen further than one high power fieldinto the tissue.

EXAMPLE 7 Monocrotaline Prevention Studies

To determine if cell-based gene transfer of VEGF165 would be capable ofinhibiting the development of pulmonary hypertension in an animal modelof disease, pulmonary artery smooth muscle cells which had beentransfected with either pVEGF or pcDNA 3.1 were trypsinized and dividedinto aliquots of 500,000 cells.

Monocrotaline is a plant alkaloid, a metabolite of which damages thepulmonary endothelium, providing an animal model of pulmonaryhypertension.

Six to eight week old Fisher 344 rats were then anesthetized andinjected subcutaneously with either 80 milligrams/kilogram ofmonocrotaline (n=13) (Aldrich Chemical Co., Milwaukee, Wis.) alone, orwith monocrotaline and, via a catheter in the external jugular vein,either 500,000 pVEGF (n=15), or pcDNA 3.1 (n=13) transfected cells. Thevein was tied off, the incision closed in the normal fashion, and theanimals allowed to recover. At 28 days after injection, animals werereanesthetized, and a Millar microtip catheter reinserted via the rightinternal jugular vein into the right ventricle. The right ventricularsystolic pressure was recorded, and the catheter was then inserted intothe ascending aorta and the systemic arterial pressure recorded. Theanimals were then sacrificed and the hearts excised. The rightventricular (RV) to left ventricular plus septal (LV) weight ratios(RV/LV ratio) were determined as an indicator of hypertrophic responseto long-standing pulmonary hypertension. Lungs were flushed via thepulmonary artery with sterile phosphate-buffered saline, and were gentlyinsufflated with 2% paraformaldehyde via the trachea. Pulmonary segmentswere then either snap frozen in liquid nitrogen for subsequent RNAextraction, or were fixed via immersion in 2% paraformaldehyde forparaffin embedding and sectioning. The right ventricular systolicpressures and RV/LV ratios were compared between the pVEGF, pcDNA 3.1,and monocrotaline alone groups.

RNA extracted from rat lungs was quantified, and 5 micrograms of totalRNA from each animal was reverse transcribed using the murine moloneyleukemia virus reverse-transcriptase, and an aliquot of the resultingcDNA was amplified with the polymerase chain reaction (PCR) using thefollowing sequence-specific primers: sense 5′ CGCTACTGGCTTATCGAAATTAATACGACTCAC 3′ (SEQ ID. NO. 3), antisense 5′ GGCCTTGGTGAGGTTTGATCCGCATAAT3′ (SEQ ID. NO. 4), for 30 cycles with an annealing temperature of 65°C. Ten microlitres of a fifty microlitre reaction were run on a 1.5%agarose gel. The upstream primer was located within the T7 priming siteof the pcDNA 3.1 vector and therefore should not anneal with anyendogenous RNA transcript, and the downstream primer was located withinexon 4 of the coding region of VEGF. Therefore, the successful PCRreaction would selectively amplify only exogenous VEGF RNA. To controlfor RNA quantity and quality, a second aliquot of the same reversetranscription reaction was amplified with the following primers for theconstitutively-expressed gene GAPDH: sense 5′ CTCTAAGGCTGTGGGCAAGGTCAT3′ (SEQ ID. NO. 5), ′, antisense 5′ GAGATCCACCACCCTGTTGCTGTA 3′ (SEQ ID.NO. 6). This reaction was carried out for 25 cycles with an annealingtemperature of 58° C. Ten microlitres of a fifty microlitre reactionwere run on a 1.5% agarose gel, and compared to the signal obtained fromthe VEGF PCR.

Paraformaldehyde fixed rat lungs were cut perpendicular to their longaxis and were paraffin-embedded en face. Sections were obtained andstained using the elastin-von Giessen's (EVG) technique. The sectionswere assessed by a blinded observer who measured all vessels with aperceptible media within each cross-section under 40× magnificationusing the C+ computer imaging system. The medial area of each vessel wasdetermined and an average was obtained for each vessel size from 0 to30, 30 to 60, 60 to 90, 90 to 120, and greater than 120 microns inexternal diameter, for each animal. The averages from each size werecompared between the pVEGF, pcDNA 3.1, and monocrotaline alone groups.

Four weeks following monocrotaline injection (n=11) alone, the rightventricular systolic pressure was increased to 48±2 mm Hg, and there wasno improvement in those animals who received the pcDNA 3.1 transfectedcells (n=10) with the average RVSP remaining at 48±2 mm Hg. However, inthose animals treated with the pVEGF transfected cells (n=15) the RVpressure was significantly decreased to 32±2 mm Hg (p<0.0001). In thisregard, see FIG. 3, which shows right ventricular systolic pressure(RVSP) graphed for the monocrotaline alone (MCT), the control vectortransfected (pcDNA 3.1) and the animals injected with the VEGFtransfected smooth muscle cells (pVEGF). Four weeks after injection ofthe pulmonary endothelial toxin monocrotaline and transfected cells, theRVSP was increased to 48 mm Hg in the MCT and pcDNA 3.1 groups, but wassignificantly decreased to 32 mm Hg in the pVEGF transfected animals.

As anticipated from the long-standing pulmonary hypertension, the RV/LVratio was significantly elevated from baseline following monocrotalineinjection (n=13) to 0.345±0.015 and was very similar in the pcDNA 3.1transfected group (n=13, 0.349±0.015, p>0.8). Following VEGF genetransfer (n=12) the ratio was significantly reduced to 0.238±0.012(p<0.0001). No difference in aortic pressure was noted. See FIG. 4, inwhich the right ventricular to left ventricular plus septal weight ratio(RV/LV ratio) is used as a measure of long-standing pulmonary and rightventricular hypertension. Four weeks after injection of the pulmonaryendothelial toxin monocrotaline and transfected cells, the RV/LV ratiois significantly elevated to 0.345 in the MCT group and 0.349 in thepcDNA 3.1 group, but was decreased to 0.238 in the pVEGF transfectedanimals.

Morphometric analysis of the tissue sections revealed that in both themonocrotaline alone and the pcDNA 3.1 treated groups, the medial areafor the vessel groups from 0 to 30, 30 to 60 and 60 to 90 microns wassignificantly increased, as compared to the VEGF treated animals(p<0.05). In this regard, see FIGS. 5A to 5C showing that four weeksfollowing subcutaneous injection of the pulmonary endothelial toxin,monocrotaline, a marked smooth muscle hypertrophic and hyperplasticresponse was observed in the mid-sized pulmonary vessels (FIG. 5A).Similar results were seen in animals transfected with the controlvector, pcDNA 3.1 (FIG. 5B). Following cell-based gene transfer of VEGF,a significant decrease in medial thickness and area was observed invessels of 0 to 90 microns external diameter (FIG. 5C). See also FIG. 6,which shows that a significant attenuation of medial area was detectedin those animals treated with monocrotaline and VEGF, as compared tothose who received monocrotaline alone or monocrotaline and the nulltransfected cells (pcDNA 3.1).

Using the viral-based primers, the exogenous VEGF transcript wasselectively amplified using the polymerase chain reaction. In thisregard, see FIG. 7 which shows that, in animals injected with the VEGFtransfected cells, a variable but consistently detectable signal couldbe detected at the correct size (lanes 1-3), however no signal wasdetectable in either the monocrotaline alone or control transfectedanimals (lanes 4 and 5). RNA quality and loading was assessed byamplifying the house-keeping gene GAPDH, which was consistently presentin all samples. This demonstrates that the foreign RNA was beingtranscribed 28 days after cell-based gene transfer and that potentiallythe presence of the transcript, and presumably the translated protein,was causally related to the lowering of RVSP in the VEGF treatedanimals.

EXAMPLE 8 Monocrotaline Reversal Studies

To determine if cell-based gene transfer of VEGF165 would be capable ofreversing or preventing the progression of established pulmonaryhypertension in an animal model of disease, six to eight week old Fisher344 rats were injected subcutaneously with 80 milligrams/kilogram ofmonocrotaline. Fourteen days after monocrotaline injection the animalswere anesthetized and a Millar catheter was passed into the rightventricle and the RV pressure recorded. Pulmonary artery smooth musclecells transfected with either pVEGF (n=10) or pcDNA 3.1 (n=8) were theninjected in aliquots of 500,000 cells into the external jugular vein,and the animals allowed to recover. At 28 days after monocrotalineinjection, and 14 days after cell-based gene transfer, the animals werereanesthetized, and a Millar microtip catheter reinserted via the rightinternal jugular vein into the right ventricle. The right ventricularsystolic pressure (RVSP) was recorded, and the catheter was theninserted into the ascending aorta and the systemic arterial pressurerecorded. The animals were then sacrificed and the hearts excised. TheRV/LV ratios were determined as an indicator of hypertrophic response tolong-standing pulmonary hypertension. The right ventricular systolicpressures and RV/LV ratios were compared between the pVEGF and pcDNA 3.1groups.

Two weeks after monocrotaline injection, the RVSP was elevated to 27±1mm Hg. In the animals who received pcDNA 3.1 transfected cells thepressure was further increased to 55±5 mm Hg at four weeks aftermonocrotaline delivery. However, in the pVEGF treated animals the RVSPhad only increased to 37±3 mm Hg (p<0.01). In this regard, see FIG. 8 inwhich the right ventricular systolic pressure (RVSP) is graphed for theanimals injected with the control vector transfected (pcDNA 3.1) and theVEGF transfected smooth muscle cells (pVEGF), 14 days aftermonocrotaline injection. Four weeks after injection of the pulmonaryendothelial toxin monocrotaline, the RVSP was increased to 55 mm Hg inthe pcDNA 3.1 group, but was significantly decreased to 37 mm Hg in thepVEGF transfected animals.

The RV/LV ratio was significantly elevated in the pcDNA group to0.395±0.022, but following VEGF gene transfer the ratio wassignificantly reduced to 0.278±0.012 (p<0.0005). Again no difference inaortic pressure was noted. In this regard, see FIG. 9, in which theright ventricular to left ventricular plus septal ratio (RV/LV) isgraphed for the animals injected with the control vector transfected(pcDNA 3.1) and the VEGF transfected smooth muscle cells (pVEGF), 14days after monocrotaline injection. Four weeks after injection ofmonocrotaline, the ratio was increased to 0.395 in the pcDNA 3.1 group,but was significantly decreased to 0.278 in the pVEGF transfectedanimals.

EXAMPLE 9 Treatment of Pulmonary Hypertension with Nitric Oxide SynthaseIntroduced by Cell Based Gene Transfer

Pulmonary artery smooth muscle cells (SMC) were harvested from Fisher344 rats, and transfected in vitro with the full-length coding sequencefor endothelial nitric oxide synthase (eNOS) under the control of theCMV enhancer/promoter. Thirteen syngenetic rats were injected with 80mg/kg of monocrotaline subcutaneously, and of these, 7 were randomizedto receive eNOS transfected SMC (5×105) via the jugular vein. 28 dayslater right ventricular (RV) pressure was measured by means of a Millarmicro-tip catheter and pulmonary histology examined.

ENOS gene transfer significantly reduced systolic RV pressure from52+/−6 mm Hg in control animals (monocrotaline alone, n=6) to 33+/−7 inthe eNOS treated animals (n=7, p=0.001). Similarly, RV diastolicpressures were reduced from 15+/−7 mm Hg in the controls, to 4+/−3 inthe eNOS treated animals (p=0.0055). In addition, there was asignificant attenuation of the vascular hypertrophy andneomuscularization of small vessels in the animals treated with eNOS.

Cell-based gene transfer of the nitric oxide synthase to the pulmonaryvasculature is thus an effective treatment strategy in the monocrotalinemodel of PPH. It offers a novel approach with possibilities for humantherapy.

Statistical Analysis

Data are presented as means±standard error of the mean. Differences inright ventricular pressures, RV/LV ratios, and medial area in the pVEGF,pcDNA 3.1, and monocrotaline transfected animals were assessed by meansof an analysis of variance (ANOVA), with a post-hoc analysis using theBonferroni correction, for the prevention experiments. Unpaired t-testswere used to compare differences in right ventricular pressures andRV/LV ratios in the pVEGF and pcDNA 3.1 treated animals, for thereversal experiments. Differences in the number of fluorescently labeledcells or transfected cells over time were assessed by means of ananalysis of variance (ANOVA), with a post-hoc analysis using a Fisher'sProtected Least Significant Difference test. In all instances, a valueof p<0.05 was accepted to denote statistical significance.

EXAMPLE 10 Skin Fibroblast Explant Culture

Fisher 344 rats (Charles River Co.) were obtained at 21 days of age andwere sacrificed by overdose with ketamine and xylazine. The hair wascarefully shaved and the back skin was excised and transferredimmediately into a phosphate-buffered saline (PBS) solution containing2% penicillamine and streptomycin (Gibco BRL, Burlington, Ontario). Theepidermal and deep fat and connective tissue was removed using ascalpel. The dermal tissue was cut into approximately 4 millimetersquare pieces which were placed on individual fibronectin-coated (SigmaChemical Co., Mississauga, Ontario) tissue culture plates (Falcon,Becton Dickinson Canada, Mississauga, Ontario). The explants were thengrown in Dulbecco's Modified Eagle Media with 20% fetal calf serum (FCS)and 2% penicillamine and streptomycin (all Gibco BRL), in a humidifiedenvironment with 95% O2 and 5% CO2 at 37° C., with the media beingchanged every second day. Explants were passaged using 0.05%trypsin/EDTA (Gibco BRL) once many thin, spindle-shaped cells couldclearly be seen growing from the dermal explant and the remainingexplanted tissue was removed. The cells were then grown in DMEM with 20%FCS and 2% penicillamine and streptomycin until they were to be used infurther experiments.

The purity of the cells as to effective type was checked, usingantibodies and standard staining techniques, to determine theapproximate number of available, effective cells of fibroblast lineage.

Fluorescent cell labeling of the cells was conducted as described inExample 1, followed by in vitro experiments to determine fluorescence,also as described in Example 1.

EXAMPLE 11 Ex Vivo Fibroblast Cell Transfection with the CMV-α GalPlasmid

The vector CMV-α Gal (Clontech Inc., Palo Alto, Calif.), which containsthe beta-galactosidase gene under the control of the cytomegalovirusenhancer/promoter sequence, was used as a reporter gene to follow thecourse of in vivo transgene expression. Fibroblasts were grown to 70 to80% confluence. The optimal ratio of liposome to DNA was determined tobe 6 μg of liposome/1 μg of DNA. Cells were washed with DMEM medium (noadditives) and 6.4 mls of DMEM was added to each 100 mm plate. 200 μl ofGenefector (Vennova Inc., Pablo Beach Fla.) was diluted in 0.8 mls ofDMEM and mixed with 16 μg of DNA (CMV-α Gal) also diluted in 0.8 ml ofDMEM. The liposome solution was then added dropwise over the entiresurface of the plate, which was gently shaken and incubated at 30° C.for eight hours. This method was used to avoid the use of viral vectorsand simultaneously obtain significant in vitro transfectionefficiencies. The Genefector product is an optimized liposomepreparation. This Genefector-DNA complex then interacts with cellsurface and is transported into the cytoplasm, after which the plasmidDNA can translocate to the nucleus.

Then the transfection medium was replaced with 20% FBS, with 2%penicillin/streptomycin in M199 media and incubated for 24 to 48 hours.This method resulted in transfection efficiencies between 40 and 60%.

EXAMPLE 12 Animal Surgery and Detection of Fluorescently-Labeled Cellsin Tissue

Animal surgery followed by introduction of dermal fibroblast cellslabeled with CMTMR or transfected with plasmid vector CMV-β Gal, wasconducted as described in Examples 4 and 5, and the fluorescentlylabeled fibroblast cells in tissue were similarly detected.

At 30 minutes or 24 hours after delivery of labeled cells (n=3 for eachtime-point), or saline injection (negative control, n=3), the animalswere sacrificed by anesthetic overdose, and the chest cavity was opened.The pulmonary artery and trachea were flushed with saline, and the rightand left lungs excised. Transverse slices were taken from the basal,medial and apical segments of both lungs, and specimens obtained fromthe liver, spleen, kidney and gastronemius muscle. Tissue specimens wereembedded in OCT compound (Sakura Finetek U.S.A. Inc., Torrance, Calif.)en face, and then flash frozen in liquid nitrogen. Ten micron sectionswere cut from these frozen blocks at 2 different tissue levels separatedby at least 200 microns, and these sections were then examined under afluorescent microscope using a rhodamine filter, and the number ofintensely fluorescing cells was counted in each en face tissue specimen.

The estimate of the total number of labeled cells present within theentire lung was obtained as described in Example 5.

Thirty minutes after fibroblast delivery, 373±36 CMTMR-labeled cells/cm2were identified within the lung sections, which representedapproximately 60% of the total number of cells injected. After 24 hoursthere was only a slight decrease in CMTMR-labeled cells to 317±4/cm2 or85% of the 30-minute value, indicating excellent survival oftransplanted cells. The survival of CMTMR-labeled cells at later timepoints of 2, 4, 7, and 14 days and 1, 2, 3 and 6 months are alsoevaluated to establish the time course of transplanted cell survival inthe lungs of recipient rats. No brightly fluorescent signals were seenin any of the lungs injected with non-labeled smooth muscle cells.

In the spleen, liver and skeletal muscle tissue no fluorescent signalswere identified. In 2 out of 4 kidneys examined, irregular fluorescentsignals could be identified. None of these appeared to conform to theshape of a whole cell, and were presumed to represent those cells thatwere sheared or destroyed during cell injection or shortly thereafter.In addition, no fluorescent signals were identified in any organ outsideof the lung 7 days after injection.

EXAMPLE 13 Monocrotaline Prevention Studies with Transfected DermalFibroblasts

The procedure of Example 7 was largely repeated to determine ifcell-based gene transfer of VEGF165 in dermal fibroblasts would becapable of inhibiting the development of pulmonary hypertension in ananimal model of the disease, dermal fibroblasts which had beentransfected with either pVEGF or pcDNA 3.1 (an empty vector) wereprepared as described above. The full-length coding sequence of VEGF165was generated by performing a reverse transcription polymerase chainreaction using total RNA extracted from human aortic smooth muscle cellsand the following sequence specific primers: sense 5′TCGGGCCTCCGAAACCATGA 3′ (SEQ ID. NO. 7), antisense 5′CCTGGTGAGAGATCTGGTTC 3′(SEQ ID. NO. 8). This generated a 649 bp fragmentwhich was cloned into the pGEM-T vector (Promega, Madison, Wis.), andsequenced to confirm identity. The fragment was then cloned into theexpression vector pcDNA 3.1 at the EcoR1 restriction site, and correctorientation determined using a differential digest. The insert deficientvector (pcDNA 3.1) was used as a control for the monocrotalineexperiments. All plasmid DNA was introduced into a JM109 strain of E.Coli via the heat-shock method of transformation, and bacteria werecultured overnight in LB media containing 100 micrograms/millilitre ofampicillin. The plasmid was then purified using an endotoxin-freepurification kit according to the manufacturer's instructions (QiagenEndotoxin-Free Maxi Kit, Qiagen Inc., Mississauga, Ontario), producingplasmid DNA with an A260/A280 ratio of greater than 1.75, and aconcentration of at least 1.0 micrograms/microliter.

Transfected dermal fibroblasts were trypsinized and divided intoaliquots of 500,000 cells. Six to eight week old Fisher 344 rats werethen anesthetized and injected subcutaneously with either 80milligrams/kilogram of monocrotaline (n=13) (Aldrich Chemical Co.,Milwaukee, Wis.) alone, or with monocrotaline and, via a catheter in theexternal jugular vein, either 500,000 pVEGF (n=5), or pcDNA 3.1 (n=3)transfected cells.

Following the procedure described in Example 7, the vein was tied off,the incision closed in the normal fashion, and the animals allowed torecover. At 28 days after injection, animals were re-anesthetized, and aMillar microtip catheter reinserted via the right internal jugular veininto the right ventricle. The right ventricular systolic pressure wasrecorded, and the catheter was then inserted into the ascending aortaand the systemic arterial pressure recorded. The animals were thensacrificed and the hearts excised. The right ventricular (RV) to leftventricular plus septal (LV) weight ratios (RV/LV ratio) were determinedas an indicator of hypertrophic response to long-standing pulmonaryhypertension. Lungs were flushed via the pulmonary artery with sterilephosphate-buffered saline, and were gently insufflated with 2%paraformaldehyde via the trachea. Pulmonary segments were then eithersnap frozen in liquid nitrogen for subsequent RNA extraction, or werefixed via immersion in 2% paraformaldehyde for paraffin embedding andsectioning. The right ventricular systolic pressures and RV/LV ratioswere compared between the pVEGF, pcDNA 3.1, and monocrotaline alonegroups.

RNA extracted from rat lungs was quantified, and 5 micrograms of totalRNA from each animal was reverse transcribed using the murine moloneyleukemia virus reverse-transcriptase, and an aliquot of the resultingcDNA was amplified with the polymerase chain reaction (PCR) using thefollowing sequence-specific primers: sense 5′ CGCTACTGGCTTATCGAAATTAATACGACTCAC 3′ (SEQ ID. NO. 9), antisense 5′ GGCCTTGGTGAGGTTTGATCCGCATAAT3′ (SEQ ID. NO. 10), for 30 cycles with an annealing temperature of 65°C. Ten microlitres of a fifty microlitre reaction were run on a 1.5%agarose gel. The upstream primer was located within the T7 priming siteof the pcDNA 3.1 vector and therefore should not anneal with anyendogenous RNA transcript, and the downstream primer was located withinexon 4 of the coding region of VEGF. Therefore, the successful PCRreaction would selectively amplify only exogenous VEGF RNA. To controlfor RNA quantity and quality, a second aliquot of the same reversetranscription reaction was amplified with the following primers for theconstitutively-expressed gene GAPDH: sense 5′ CTCTAAGGCTGTGGGCAAGGTCAT3′ (SEQ ID. NO. 11), antisense 5′ GAGATCCACCACCCTGTTGCTGTA 3′ (SEQ ID.NO. 12). This reaction was carried out for 25 cycles with an annealingtemperature of 58° C. Ten microlitres of a fifty microlitre reactionwere run on a 1.5% agarose gel, and compared to the signal obtained fromthe VEGF PCR.

Paraformaldehyde fixed rat lungs were cut perpendicular to their longaxis and were paraffin-embedded en face. Sections were obtained andstained using the elastin-von Giessen's (EVG) technique. The sectionswere assessed by a blinded observer who measured all vessels with aperceptible media within each cross-section under 40× magnificationusing the C+ computer imaging system. The medial area of each vessel wasdetermined and an average was obtained for each vessel size from 0 to30, 30 to 60, 60 to 90, 90 to 120, and greater than 120 microns inexternal diameter, for each animal. The averages from each size werecompared between the pVEGF, pcDNA 3.1, and monocrotaline alone groups.

Four weeks following monocrotaline injection (n=11) alone, the rightventricular systolic pressure was increased to 48±2 mm Hg, and there wasno improvement in those animals who received the pcDNA 3.1 transfectedcells (n=3) with the average RVSP remaining at 48±2 mm Hg. However, inthose animals treated with the pVEGF transfected fibroblasts (n=5) theRV pressure was significantly decreased to 32±2 mm Hg (p<0.0001).

As anticipated from the long-standing pulmonary hypertension, the RV/LVratio was significantly elevated from baseline following monocrotalineinjection (n=13) to 0.345±0.015 and was very similar in the pcDNA 3.1transfected group (n=3). Following VEGF gene transfer (n=5) the ratiowas significantly lower than the pcDNA 3.1 transfected group. Nodifference in aortic pressure was noted. Four weeks after injection ofthe pulmonary endothelial toxin monocrotaline and transfected cells, theRV/LV ratio is significantly elevated in the MCT group and in the pcDNA3.1 group, but was lower in the pVEGF transfected animals.

Morphometric analysis of the tissue sections revealed that in both themonocrotaline alone and the pcDNA 3.1 treated groups, the medial areafor the vessel groups from 0 to 30, 30 to 60 and 60 to 90 microns wassignificantly increased, as compared to the VEGF treated animals.Similar results were seen in animals transfected with the controlvector, pcDNA 3.1. Following cell-based gene transfer of VEGF, asignificant decrease in medial thickness and area was observed invessels of 0 to 90 microns external diameter.

Using the plasmid-based primers, the exogenous VEGF transcript wasselectively amplified using the polymerase chain reaction. RNA qualityand loading was assessed by amplifying the house-keeping gene GAPDH,which was consistently present in all samples. This demonstrated thatthe foreign RNA was being transcribed 28 days after cell-based genetransfer and that potentially the presence of the transcript, andpresumably the translated protein, was causally related to the loweringof RVSP in the VEGF treated animals.

Blood taken from the animals by left ventricular puncture immediatelybefore sacrifice was analyzed for pH, oxygen loading (pO2), carbondioxide loading (pCO2) and % saturation. The results are given below.

BLOOD ANALYSIS DATA VEGF/fibroblast transfected animals pH pCO2 pO2 %Sat'n Mean (of 5 animals) 7.374 51.2 78.3 86.58 Standard deviation, SD0.0502 5.699 17.89 9.867 pcDNA/fibroblast transfected animals pH pCO2pO2 % Sat'n Mean (of 3 animals) 7.35 58.333 60.8 74.4 SD 0.02 3.7617.615 7.882

These results indicate preliminarily that arterial O2 tension andsaturation are better in the VEGF transfected group than in animalsreceiving null-transfected cells. This is consistent with theimprovement in pulmonary hemodynamics and lung vascular morphology, andargues against significant right to left shifting as might occur inpulmonary arterial to venous shunts.

The creation of “shunting”—formation of new passageways between thearteries and the capillaries of the pulmonary system, by-passing theveins and thereby limiting the blood oxygen up-take, does not occur toany problematic extent, according to indications.

DISCUSSION

The present invention represents evidence of successful non-viral genetransfer to the pulmonary vasculature using various types of transfectedcells e.g. smooth muscle cells and dermal fibroblasts, and provides ademonstration of potential therapeutic efficacy of an angiogenicstrategy in the treatment of PH using this approach. This method ofdelivery was associated with a high percentage of cells being retainedwithin the lung at 48 hours, as determined by both the fluorescencelabeling technique and by the reporter gene studies usingbeta-galactosidase, and with moderate but persistent gene expressionover 14 days. These results roughly parallel what has previously beendemonstrated with a viral-based method of intravascular gene delivery tothe pulmonary vasculature (see Schachtner, S. K., J. J. Rome, R. F.Hoyt, Jr., K. D. Newman, R. Virmani, D. A. Dichek, 1995. In vivoadenovirus-mediated gene transfer via the pulmonary artery of rats.Circ. Res. 76:701-709; and Rodman, D. M., H. San, R. Simari, D. Stephan,F. Tanner, Z. Yang, G. J. Nabel, E. G. Nabel, 1997. In vivo genedelivery to the pulmonary circulation in rats: transgene distributionand vascular inflammatory response. Am. J. Respir. Cell Mol. Biol.16:640-649).

However, the cell-based technique provided by the present inventionavoids the use of a potentially immunogenic viral construct, was notassociated with any significant pulmonary or systemic inflammation, andpermits more selective transgene expression within the pulmonarymicrovasculature, and in particular the targeting of transgeneexpression localized to the distal pulmonary arteriolar region, which isprimarily responsible for determining pulmonary vascular resistance, andtherefore PH.

The present invention addresses several key questions related to thefeasibility of a cell-based gene transfer approach for the pulmonarycirculation, including the survival of genetically engineered cells andthe selectivity of their localization and transgene expression withinthe lungs. As demonstrated above in Example 6, implanted cells wereefficiently retained by the lungs.

The finding that most of the cells appeared to lodge within smallpulmonary arterioles is consistent with the normal physiological rolethe lung plays as an anatomical filter, and thus it would be expectedthat relatively large particles such as resuspended cells would becomelodged within the pulmonary microvasculature. However, this ‘targeting’of cells to the pre-capillary resistance vessel bed in a highlyselective manner may prove very useful in the treatment for certainpulmonary vascular disorders. The overexpression of a vasoactive gene atthe distal arteriolar level could provide a highly localized effect in avascular region critical in the control of pulmonary vascular resistanceand could amplify the biological consequences of gene transfer. In fact,the localized reduction in RVSP seen in monocrotaline-treated animalsreceiving VEGF transfected cells, occurred without a correspondingdecrease in systemic pressures, highlighting the specificity of thismethod of transfection. This approach may therefore offer significantadvantages over other pulmonary selective gene transfer strategies suchas endotracheal gene delivery, which results in predominantly bronchialoverexpression, or catheter-based pulmonary vascular gene transfer,which produces diffuse macrovascular and systemic overexpression (seeRodman, D. M., H. San, R. Simari, D. Stephan, F. Tanner, Z. Yang, G. J.Nabel, E. G. Nabel, 1997. In vivo gene delivery to the pulmonarycirculation in rats: transgene distribution and vascular inflammatoryresponse. Am. J. Respir. Cell Mol. Biol. 16:640-649; and Nabel, E. G.,Z. Yang, D. Muller, A. E. Chang, X. Gao, L. Huang, K. J. Cho, G. J.Nabel, 1994. Safety and toxicity of catheter gene delivery to thepulmonary vasculature in a patient with metastatic melanoma. Hum. GeneTher. 5:1089-1094).

This significant effect occurred despite an overall relatively low massof organ-specific transfection, and was likely due to the fact that thetransfected cells were targeted, based on their size, to theprecapillary pulmonary resistance vessels which play a critical role incontrolling pulmonary pressure. This method of pulmonary vascular genetransfer may have benefits over existing techniques by minimizing theoverall “load” of foreign transgene that is delivered to the body andmay thereby theoretically reduce the incidence of undesiredside-effects.

EXAMPLE 14 Ex Vivo Smooth Muscle Cell Transfection with ProstacyclinSynthase

Endothelial cell (EC) injury and dysfunction is believed to be an earlyevent in PPH. Activation of endothelial cells has been found in diverseanimal models of PH, including the rat chronic hypoxia and monocrotaline(MCT) models, as well PH induced by endotoxin, diaphragmatic hernia, orair-induced chronic pulmonary hypertension. EC dysfunction may in turngive rise to an imbalance of vasodilatory and vasoconstrictive agents.An altered ratio of thromboxane to prostaglandin and increased plasmaendothelin-1 (ET-1) levels have all been reported in PPH. It is wellrecognized that there is a pathological remodeling of the pulmonaryvasculature, characterized by intimal fibrosis, medial hypertrophy, andadventitial proliferation in late stage. Administration of PGI₂analogues has been shown to result in the effective treatment of PPH ina number of clinical studies. Therefore, the rate limiting enzyme in thepathway for PGI₂biosynthesis, prostaglandin I synthase (PGIS) isattractive target for cell-based gene therapy.

PGIS Gene Therapy in Experimental PH:

Prostacyclin synthase (PGIS), is a key enzyme involved in the productionof prostacyclin, catalyzing the conversion of PGH₂ to PGI₂(prostacyclin), a potent vessel dilator and cell growth inhibitor. Thisenzyme has also been shown to be downregulated in patients with severePH. Experiments in PH animal models have demonstrated that PGIS canprotect against the development PH, and slow its progression, suggestingthat PGIS is a promising agent for treatment of pulmonary hypertension.Clinical studies using intravenous infusion (Flolan), subcutaneousinjection (Remodulin) or inhalation (Iloprost) have all reported benefitin patients with PAH.

The objective was to clone the full-length human PGIS cDNA and test itsproduction activity.

Cloning and Verification of Activity of hPGIS:

RT-PCR was used to amplify the hPGIS cDNA from a human smooth musclelibrary. Primers QW9 and QW18 were designed to yield a full-length cDNAproduct, with an expected size of 1.5 kb (see FIG. 10), whichcorresponds to the size of hPGIS cDNA. Primers QW18 and QW9, innerprimers, are used for the second-stage of PCR to amplify hPGIS cDNA.

QW9: 5′-CGA GCA CGT  GGA TCC  ATC-3′  (SEQ ID. NO. 13; antisense or PGIS cDNA, position  1532-1515; Tm =58 (BamH I site underlined) QW18: 5′-CAT GGA TCC GCG ATG G CT TGG GCC-3′(SEQ ID. NO. 14; sense, for cloning hPGIS cDNA, position -5---12; Tm =60)(BamH I site underlined)

FIG. 10 shows a band of 1.5 kb (arrowhead) was amplified (lanes 1 and2). Lane 3 is a DNA size marker.

The 1.5 kb fragment was isolated and cloned into the pVAX1 vector. Theresulting plasmid grown in competent E. Coli and purified by Maxiprep.The insert was released by restriction enzyme digestion and sequenced.One clone was shown to be 100% homologous with hPGIS with the kozaksequence immediately upstream of the start codon (ATG), and a stop codonat position 1500. Transfection of hPGIS cDNA in COS-1 cells: The hPGIScDNA was successfully expressed in COS-1 cells with a molecular weightof about 50 kD. Biological activity of hPGIS: 6-keto PGF1 alpha, thestable metobolite of PGI2, was detected by ELISA in conditioned mediumof human SMCs transfected with hPGIS in two different experiments (seetable). In both assays, transfected cells produced about 2-3-foldgreater levels of PGI2 than control (mock) transfected cells.

6-keto PGF2-alpha levels in HASMCs in 2% FCS (pg/ml) N pVAX-1 pVAX-hPGIS4 h 772 1533 8 h 1782 3778 6-keto PGF2-α levels in HASMCs in 2% FCS(pg/ml)

EXAMPLE 15 Monocrotaline Studies with Smooth Muscle Cells Transfectedwith PGIS

The objective was to test the efficacy of cell-based gene therapy withhuman PGIS in the rat MCT model in comparison with eNOS and VEGF.

FIGS. 11 and 12 show cell-based gene transfer using PGIS in experimentalpulmonary hypertension (prevention protocol).

An experiment was completed testing the effect of cell based genetherapy using hPGIS (n=6) and eNOS (n=6) compared with null transfectedanimals (n=7). Gene therapy was given together with MCT (70 mg/kg) andall animals received a total of 1.5 million cells in 3 divided doses.Unfortunately, the mortality rate was higher than expected in the PGISgroup likely due to biological variation in the sensitivity of thisbatch of rats (2/6 for PGIS; 0/6 for eNOS and 0/7 for null). Thehemodynamic data for animals surviving until end-study are presented inFIGS. 11 and 12. Animals receiving MCT together with null transfectedfibroblasts (FBs) exhibited elevated RVSP, indicative of PH (47.8±2.2mmHg). In the MCT-treated rats which received 3 doses ofPGIS-transfected FBs, RVSP was reduced to 36.6±0.263 mmHg, and thebenefit appeared similar in this series to rats treated with eNOS genetransfer. The RV/LV was 0.3 in MCT-treated group, compared to 0.28 ingroup received three dosing of PGIS (RV/LV in normal rat is 0.23) (seeFIG. 12).

CONCLUSIONS

PGIS gene transfer may improve pulmonary hemodynamics in experimental PHto a degree similar to that seen with eNOS.

EXAMPLE 16 Cell Based Gene Therapy in Established Pulmonary HypertensionUsing Reversal Protocol

The present inventor has demonstrated that cell-based gene therapy canprevent monocrotaline (MCT) induced pulmonary hypertension using theVEGF and eNOS transgene. The efficacy cell-based gene therapy wasassessed in experimental models of established PH, so that the abilityof this treatment to reverse structural and functional abnormalities ofthe pulmonary circulation could be shown.

Objectives:

To study the efficacy of cell-based gene transfer to reverse establishedPH in the MCT model.

Methods and Results:

These studies employed a modification of the standard MCT experimentalprotocol previously validated with experiments using eNOS gene transferand VEGF gene transfer in examples set out above. Briefly, MCT wasinjected as usual, and at day 21 post MCT the animals were thenanaesthetized and RVSP was recorded. Rats are then randomly assigned asnormal (n=40), to receive null-transfected (pcDNA, n=32) or cellstransfected with an active transgene (VEGF, n=20; eNOS, n=36), and thensurvived until day 35, at which time RVSP is remeasured, and the ratsare sacrificed morphometric, functional and molecular assessments. Inthe present studies, human VEGF₁₆₅ was used since it was hypothesizedthat reversal of PH must involve regeneration of occluded pulmonaryarterioles. Again, animals were treated with a total of 1.5 millioncells, delivered in 3 divided doses.

As shown in FIG. 13, at day 21 (i.e. prior to gene therapy), RVSP wassimilarly elevated in the control (null) and VEGF and eNOS transfectedgroups (approximately 40 mm Hg). In the MCT treated with nulltransfected FBs, there was a further significant increase in RVSP at day35 (51.6±4 mmHg, p<0.05), indicating progression of PH. In theMCT-treated rats receiving VEGF transfected FBs, RVSP demonstrate atrend towards reduction as compared to the null transfected FBs. In theMCT-treated rats receiving eNOS transfected FBs, RVSP demonstrate atrend towards significant reduction (p<0.05 vs. normal). RV/LV wasincreased in the MCT-null vector group (0.29), however this was reducedto 0.27 in the group receiving VEGF (RV/LV in normal rat is 0.25). Theweight gain is 84 g in normal group and reduced to 48 g in MCT treatedgroup. Weight gain tended to increase to 58 g in the VEGF treated group.

CONCLUSIONS

In this series, cell-based gene therapy VEGF prevented furtherprogression of established PH. Cell based GT with NOS effectivelyreversed hemodynamic abnormalities in established PH and resulted in theregeneration of continuity of the pulmonary microcirculation.

EXAMPLE 17 Optimization of Nonviral Transfection Efficiency forFibroblasts

This Example sets out the utility of sequential transfection withb-cationic proteins (Superfect).

Objective:

To establish a standard operating protocol (SOP) for optimal transienttransfection of rat and human FBs using nonviral methods of genetransfer and sequential gene transfer.

Methodology:

i) Cell preparation: Rat FBs were plated 12-24 hours prior totransfection resulting in a confluence of 60-80%. For 35 mm plates usedfor practice transfection that is 50,000 cells, T-75 is 1,000,000 cells.Growths conditions are DMEM (Gibco, #119950065) containing 15% serum(Sigma, F-2442) at 37° C. in 5% CO₂

ii) Transfection protocol: the following were mixed in a 50 ml Falcontube (falcon, cat. #352070):

a) 500 ul DMEM medium, no serum or anti-biotic

b) 7 ug plasmid DNA, i.e. vector plus insert.

c) 50 ul superfect (Qiagen, cat #301307, 3 mg/ml)

The mixture was then added to each T-75 flask (superfect/DNA complex).

Superfect/DNA complex is incubated for 5-10 minutes upon which added 5ml of DMEM containing 15% serum and added to the cell population for 5-8hours.

In “double transfection” protocols, cells that have been alreadytransfected, are re-plated as described above and re-transfected 48hours after the first transfection procedure. This time interval hasbeen determine to be optimal in studies (data not shown). In the“triple”transfection” protocol, a third transfection is performed again after a48 hour “recovery” interval”. This approach has the theoreticaladvantage of allowing transfection of a separate population of cellsfrom those susceptible in the first transfection, while avoidingsignificant toxicity which would otherwise occur.

Measurements:

To determine the cell number and DNA/superfect ratios that give the bestresults, two methods have been selected:

i) RT-PCR, genes selected for measurement,

ii) VEGF165, B-gal, eNOS and the house keeping gene GAPDH.

Primers for these genes are:

Exogenous VEGF: VHF1  5′-cgc tac tgg ctt atc gaa att aat acg act cac,VHF2  5′-ggc ctt ggt gag gtt tga tcc gca taa t; exogenous eNOS:VHF1, NHR ® 5′-cgc tct ccc taa gct ggt agg tgc c; β-gal: β-gal (1) 5′- tgt acc cgc ggc cgc aat tcc, β-gal (2) 5′- att cgc gct tgg cct tcc tgt agc c; GAPDH: GDH1   5′ctc taa ggc tgt ggg caa ggt cat, GDH2 5′-gag atc cac cac cct gtt gct gta.

ii) β-gal staining was used to determine best results for percent ofcells transfected.

Results:

FIG. 14 shows the results of multiple transfections using the cDNA foreNOS.

There is a near linear increase in transfection efficiency with eachsequential transfection, whereas cell viability is not reduced.

In FIG. 14, the hVEGF expression by PCR is shown after a singletransfection protocol, contrasting the effect of different superfect toDNA ratios on transfection efficiency: lane 1, non-transfected cells;lane 2, 1 μg DNA: 10 μl superfect; lane 3, 2 μg DNA: 10 μl superfect;and finally lane 4, 3 μg DNA: 10 μl superfect. Keeping the superfectconstant and increasing the amount of DNA appears to yield a largersignal.

β-gal staining is shown in FIG. 15 comparing a single transfection to adouble protocol. Double transfection results in about twice the numberof cells staining positive with LacZ.

CONCLUSIONS

Sequential transfection using ?-cationic proteins (Superfect) resultedin a near linear increase in transfection efficiency, measured both bynumber of cells expressing a reporter gene (LacZ) and amount of plasmidDNA (quantitative PCR), without an increase in toxicity.

EXAMPLE 18 Cell-Based Gene Transfer for Cystic Fibrosis Introduction andRationale:

Cystic fibrosis is an autosomal recessive disorder caused by theproduction of a defective chloride channel, CFTR, primarily expressed inepithelial cells and submucosal gland cells, and affecting multipleorgans. This genetic defect impairs transepithelial salt transport,mucous viscosity, and ion flux in organs such as the salivary glands,pancreas, gastrointestinal tract, reproductive tract, and mostimportantly, the lungs. The defect in the pulmonary epithelium resultsin highly viscous mucous, causing plugging of the tracheobronchial tree,thus interfering with normal respiratory function and increasingsusceptibility to lung infections. Our laboratory has developed a noveland highly selective method for targeting gene transfer to the pulmonaryvasculature using transfected smooth muscle cells or fibroblastsinjected via the systemic circulation. We have shown that these cellsare efficiently filtered by the distal arteriolar (pre-capillary) bedand rapidly translocate through the endothelial basement membrane totake up residence within the perivascular space. Therefore, it appearsthat these cells are able to recognize their appropriate location withinthe lung tissue.

The present inventor has found that pulmonary cell types are able to“home” to their appropriate tissue locations, possibly by recognizingspecific matrix components through unique integrin interactions. In thepresent research project, it is shown that injected pulmonary alveolartype II cells can translocate through the vascular and epithelialbasement membranes and localize to the luminal side of the epithelialbasement membrane. This would then enables transvascular delivery ofgenetically engineered epithelial cells useful in treating geneticdisorders of airway function, such as cystic fibrosis, which can then betested in a CFTR knockout mouse model.

Hypothesis:

Isolated epithelial cells from the lungs of syngeneic rats will migrateto a bronchial/bronchiolar location of normal rats when injected intothe pulmonary circulation.

Objectives:

1. To establish a primary cell culture of lung epithelial cells obtainedfrom syngeneic Fisher-344 rats.

2. To follow the in vivo migration of transplanted, CMTMR-labeledepithelial cells upon delivery into the pulmonary bed through the rightexternal jugular vein. The presence of these cells and their locationwill be evaluated over a period of one week, with rats sacrificed at 1,2, 3, and 7 days.

3. To assess the ability of transfected epithelial cells to express areporter transgene in situ after grafting into the tracheobronchialsystem.

Results:

Transplanted epithelial cells can indeed migrate to theirbronchioalveolar location.

The results are summarized in the FIG. 16 which indicates the morphologyof isolated lung epithelial cells in primary cell culture, 5 days afterisolation. Right-hand panel shows transfection of the isolated lung“epithelial” cells with β-Gal. FIG. 17 shows fluorescent microscopyshowing purity of isolated lung epithelial cells.

Green indicates positive staining for the type II epithelial markerSPAn; Blue represents nuclear staining with Dapi.

Unlike traditional virally-based gene therapy, a cell-based gene therapyapproach is less likely to provoke an immunological response andlowers/eliminates the risk of insertional mutagenesis.

EXAMPLE 19 Cell-Based Gene Transfer in Adult Respiratory DistressSyndrome (ARDS)

The angiopoietin system appears to play a critical role in themaintenance of normal endothelial homeostasis, in part by reducingendothelial permeability and preventing extravasation of plasmaproteins. Acute lung injury caused by a wide variety of insults(including ventilation-induced lung injury) results in increasedpulmonary capillary permeability and pulmonary edema without anyincrease in capillary or left atrial pressures: so called “low pressureedema” or ARDS. This is the single most common pulmonary complication ofICU patients, and accounts for a tremendous burden of morbidity andmortality. Angiopoietin-1 is a recently identified ligand of theendothelium-specific tyrosine kinase receptor Tie-2. It is involved inthe maturation of blood vessel and is very potent in reducing their thehyper-permeability response to inflammatory stimuli.

Cell based gene transfer with Angiopoietin-1 (Ang-1) reduced lung edemain an animal model of ARDS. The Angiopoietin-1 gene was introduced intorats prior to exposure to either LPS (which serves as model for sepsis)or high volume mechanical ventilation. Both of these stimulations wouldnormally be expected to induce pulmonary edema. It is involved in theangiogenic phase of embryonic vascular development with major defects inthe interaction of endothelial cells, with the surrounding mesenchymalcells and extracellular matrix evident in Ang1 knockout mice.

Objectives:

The main objectives were (1) to show that transfer of a gene(angiopoietin-1) using a cell-based transfer system could reduce theformation of pulmonary edema that occurs with the systemic inflammationresponse induced by administration of mechanical ventilation and (2) toshow that this method of gene delivery was suitable to treat disorderswhich diffusely affect the alveoli and/or capillaries in the lung.

Methods:

Preparation of the Cells Transfected with Angiopoeitin 1 Gene:

21 day old Fisher 344 rats were sacrificed by overdose of IP injectionof pentobarbital (50 mg). The pulmonary artery was dissected out, andsmooth muscle cells were cultured and transfected with the genefollowing the established protocol.

Intravenous Delivery of Transfected Cells, Untransfected Cell or NormalSaline:

Fisher 344 rats (body weight 200-250 gram) were anesthetized with IPxylazine (5 mg/kg) and ketamine (70 mg/kg). A midline cervical incisionwas made after cleaning and shaving the area, and the common andexternal jugular veins identified. Animals were randomized to receivedAngiopoeitin 1, untransfected cell or normal saline. Using a 23-gaugeneedle, a 1 mm tube was introduced into the external jugular vein, andthrough this approximately 500000 cells transfected with theAngiopoeitin 1 gene, untransfected cell (pcDNA3.1) as a control groupand 1 cc normal saline as a sham group were infused. The animals wereallowed to recover for 24 hours.

Induction of Pulmonary Edema:

The rats were mechanically ventilated in order to stimulate pulmonaryedema. The rats were anesthetized with ketamine (75 mg/kg) and xylazine(15 mg/kg). A mid-cervical incision was made, the trachea exposed andincised. A 16 G catheter was inserted into the trachea, through whichthe animal was ventilated. The tail vein was cannulated, and an IVinfusion of ketamine (20 mg/hr) xylazine (2 mg/hr) and the musclerelaxant, pancuronium (0.2 mg/hr) was commenced. The pancuronium isnecessary in order to suppress any spontaneous respiratory effort thatmight interfere with the function of the ventilator. Mechanicalventilation was commenced, using a rodent ventilator, with room air,tidal volume 20 ml/kg, zero positive end expiratory pressure andrespiration rate 27/bpm. The carotid artery was cannulated with 24 Gangiocatheter and connected to BP monitor with a three-way stock. Werecorded the mean artery pressure, peak airway pressure, plateau airwaypressure and measured the artery blood gas at baseline, 0.5, 1, 2 and 3hour during the ventilation. After 3 hours ventilation, the animals weresacrificed by IV injection of pentobarbital, and the lungs processed asabove to obtain the wet/dry weight ratio.

Results

(A) Healthy Lung Model: Baseline 1 hour 2 hour 3 hour Ang 112     121   112 110     Con (pc DNA)  97.66667 103.3333  93  86.66667 * Mean arterypressure (mm Hg) change during the ventilation Baseline 1 hour 2 hour 3hour Ang 21.16667 19.93333 21.16667 21.6   Con (pc DNA) 22.2666722.86667 22.66667 22.06667 * Peak airway pressure (cm H2O) change duringthe ventilation Baseline 1 hour 2 hour 3 hour Ang 17.33333 16.6666717.83333 18     Con (pc DNA) 18.33333 18.83333 18.83333 18.16667 *Plateau airway pressure (cm H2O) change during the ventilation Body WetDry W/D Weight Weight Weight Weight (gm) (gm) (gm) ratio (gm) Ang 240   1.029   0.222   4.635135 Ang 240    1.07   0.212   5.04717  Ang 227   1.077   0.215   5.009302 Mean Value 235.6667 1.058667 0.216333 4.897202Con (pc DNA) 243    1.115   0.226   4.933628 Con (pc DNA) 236    1.226  0.217   5.64977  Con (pc DNA) 235    1.268   0.225   5.635556 Mean Value238    1.203   0.222667 5.406318 n = 3 in both group no significantdifference except trend in W/D ratio in both group (p value = 0.1329)

FIGS. 18 to 20 show a summary of the results of Ang-1 gene therapy forARDS using the ventilator induced lung injury model. FIG. 18 shows asignificant decrease in Wet/Dry lung weight by use of gene therapy. FIG.19 shows a significant decrease in peak airway pressure by use of genetherapy. FIG. 20 shows maintenance of partial oxygen pressure ascompared to the null vector, by use of gene therapy.

As shown in the tables above, and in FIGS. 18 to 20, there was reducedlung wet to dry weight ration in animals receiving Ang-1 gene therapy,consistent with a reduction in permeability. Thus this treatmentapproach reduces pulmonary edema and capillary permeability in thismodel of ARDS.

EXAMPLE 20 Cell Based Gene Therapy in Established Pulmonary HypertensionUsing Multiple Injections

The objective was to test the efficacy of multiple injections ofcell-based gene therapy with eNOS.

An experiment was completed testing the effect of cell based genetherapy using eNOS (n=6) compared with null transfected animals (n=11)and normal animals (n=5). Gene therapy was given together with MCT (70mg/kg) and all animals received a total of 1.5 million cells in 3divided doses.

FIGS. 21 to 23 show that dosing cell-based endothelial NOS gene transferinhibits MCT-induced PH and that the effect of multiple dosing isgreater than the effect of single dosing, whether measured by RVSP (FIG.21), RV/LV+S (FIG. 22), or weight gain (FIG. 23). These results indicatethat multiple injections of eNOS-transfected cells show a dose-dependentreduction in pulmonary blood pressure, preventing hemodynamicabnormalities in PH model.

CONCLUSIONS

eNOS gene transfer may improve pulmonary hemodynamics in experimental PHin both single and multiple dosage regimens.

EXAMPLE 21 Effect of ELPC Transplantation and e-NOS-Transduced ELPCTransplantation of Pulmonary Arterial Hypertension (PAH) Objectives

The objective was to test the efficacy of ELPC cells, and cell-basedgene transfer using eNOS-transduced ELPC cells, to reverse establishedPH and to prevent PH in the MCT model.

Methods: (a) Isolation and Culture of ELPC Cells

Skin biopsies were obtained from 21-day-old Fisher-344 rats (CharlesRiver Co. St Constant, Quebec, Canada), and FBs were cultured using anexplant technique. Cells were grown in Dulbecco Modified Eagle Media(DMEM) with 10% fetal bovine serum (FBS) and 2% penicillin/streptomycin(50 U/mL penicillin G; 50 μg/mL streptomycin) in a humidified incubator(20% O₂, 5% CO₂ at 37° C.), and used between passages 2 and 9.

Bone marrow (BM) was aspirated from the femurs of 21-day-old syngeneicFisher-344 rats. Mononuclear cells (MNCs) were isolated by densitygradient (Ficoll-Paque, Amersham) centrifugation at 400 g for 30minutes. BM-MNCs were resuspended in differential endothelial cellculture medium (EBM-2, Cambrex) with 10% FBS, 50 U/mL penicillin, 50μg/mL streptomycin, and 2 mmol/L L-glutamine (Invitrogen), plated ongelatin-coated tissue culture flasks and incubated at 37° C. with 5% CO₂for 7 to 10 days, to produce endothelial-like progenitor cells (ELPCs).

For immunocytochemistry, differentiated MNCs were subcultured on 4-wellchamber slides (BD Bioscience), and fixed in 2% paraformaldehyde for 10minutes. Cells were incubated overnight at 4° C. with the followingprimary antibodies: rabbit anti-human Flk-1 (VEGF-R2; Alpha DiagnosticInc; 1:200); mouse anti-human Tie-2 (Upstate Biotechnology Inc; 1:50),or rabbit anti-human von Willebrand factor (vWF, DAKO; 1:1000). Rabbitanti-mouse or goat anti-rabbit F(ab′)2 (Vector; 1:150) conjugated withFITC were used as secondary antibodies, as appropriate. Surface lectinstaining was performed using fluorescently labeled UEA-1 Lectin (Sigma)at 10 ug/mL. As well, the ability for live cells to take upfluorescently labeled acetylated-LDL (Dil-Ac-LDL; Molecular Probes) wasassessed by incubation with Di-ac-LDL (10 ug/mL) for 4 hours at 37° C.ToPro3 (1:1000; Molecular Probes) was used for nuclear counterstainingand images were captured by confocal microscopy (BioRad Radiance).

(b) Transduction

The full-length coding sequence of human eNOS was generated aspreviously described, and ELPCs were transduced with human eNOS clonedinto the pVax-1 plasmid vector using electroporation (MaxCyte) accordingto a protocol optimized by the manufacturer. The empty (null) pcDNA 3.1vector was used as a control. After transfection, cells were replatedand cultured for 24 hours, trypsinized (0.25% trypsin, 1% EDTA), washed,and resuspended in phosphate buffered saline (PBS), and then dividedinto aliquots of 500 000 cells/mL for injection. Western blot analysisrevealed that electroporation resulted in peak human transgeneexpression at 72 hours, persisting for more than 1 week (data notshown).

(c) Animal Models of PAH

Two complementary models of MCT-induced PAH were used in this study, toexamine the effect of ELPC delivery on both prevention and reversal. Allanimal studies were conducted under protocols approved by the animalcare committee at St Michael's Hospital and in accordance withguidelines from the Canadian Council of Animal Care. In the preventionprotocol, cell therapy was delivered 3 days after MCT injection and ratswere followed for 21 days; control groups were compared with theELPC-treated group. Persistence of therapeutic effect was investigatedin separate groups of animals survived for longer periods (dottedlines). For reversal studies, rats were randomized to treatment groupsat 21 days after MCT injection and initial hemodynamic measurements weremade to confirm establishment of pulmonary arterial hypertension and toallow for paired comparisons within groups at end study 14 days later.

(d) Prevention Protocol

Cells were delivered via central venous injection 3 days after MCT, andthe animals were euthanized at 21 days. Six-week-old Fisher-344 rats(160 to 180 g) were given intraperitoneal (IP) injections of saline(control group, n=13) or 75 mg/kg of MCT (Aldrich Chemical Co). Threedays later, MCT-treated animals were assigned to three experimentalgroups: no cell injection (MCT alone, n=15), or 1 million ELPCs (n=23)or FBs (n=10). For cell delivery, rats were anesthetized with an IPinjection of xylazine (4.6 mg/kg) and ketamine (7 mg/kg), the leftcervical area was shaved and cleaned with 70% ethanol, and the externaljugular vein was catheterized with a polyethylene cannula flushed withheparinized saline (40 IU/mL). Twenty one days after MCT injection, therats were reanesthetized, and a 3F Millar microtip catheter was insertedvia the right external jugular vein and into the right ventricle toobtain measurements of right ventricular systolic pressure (RVSP; BiopacSystem, Acknowledge software). The animals were then euthanized and thehearts and lungs harvested. The ratio of right to left ventricular plusseptal weight (RV/LV) was determined as described previously. The leftlung was inflated with OCT (Tissue-Tek) and cut into pieces that werefixed in a 4% paraformaldehyde/0.1% glutaradehyde PBS solution forparaffin embedding and sectioning. The right lung was snap frozen inliquid nitrogen.

In a separate experiment, animals were treated with MCT and randomizedat 3 days to receive either no cells (saline, n=13) or ELPCs (n=12) asdescribed above and then followed for longer periods of time toestablish the persistence of any therapeutic effect. The animals weremonitored daily by experienced animal care personnel in a blindedfashion and euthanized if predetermined criteria of significantmorbidity were met (weight loss, hunched posture, poor coat appearance,conjunctival hemorrhage, and labored breathing). RVSP and RV/LV weightratios were measured at the time of euthanasia as described above.

(e) Reversal Protocol

In the reversal model, rats were injected with saline (Control, n=12) orMCT, and 21 days later, baseline RVSP was recorded as earlier to confirmthe presence of PAH. Thereafter, polyethylene catheters were insertedinto the left external jugular vein and tunneled subcutaneously to theintrascapular region, exiting through a small incision, and sealed tothe external environment with a removable plug. All incisions wereclosed with 3-0 interrupted absorbable sutures. Rats were randomized toreceive saline (MCT alone), ELPCs alone, or ELPCs transduced with humaneNOS (n=19 to 23/group). Cells were given in three sequential injectionsof 5×10⁵ over 3 days through the indwelling catheter (total dose=1.5×10⁶cells). After the final cell injection, the indwelling catheter wasremoved, the left external jugular vein was ligated, and animals wereallowed to recover. Fourteen days later (35 days after MCI) RVSP wasrecorded, the animals were euthanized, and lung and heart tissues werecollected for analyses as described.

(f) Fluorescent Microangiography

In a subset of animals, a catheter was inserted into the pulmonaryartery immediately after euthanasia and the lungs flushed withheparinized PBS at 37° C., immediately followed by perfusion with awarmed (45° C.) solution of 1% low melting point agarose (Sigma)containing 0.2 μm yellow-green fluorescent microspheres (505/515 nm peakexcitation and emission, Molecular Probes).

(g) Engraftment of ELPCs

In separate experiments, ELPCs were loaded with the vital cytoplasmicfluorescent label, CMTMR (Molecular Probes). Before transplantation,subconfluent cultures of ELPCs were incubated for 40 minutes with 10μmol/L CMTMR, and 1×10⁶ labelled cells were injected into the pulmonarycirculation of normal rats at 3 or 21 days after MCT injection via theexternal jugular vein as described. Lungs or kidneys were harvested atvarious time points (10 minutes to 3 weeks) after cell delivery andexamined by confocal fluorescent microscopy. Quantitation of cell numberwas performed. In some cases, the lungs were also subjected tofluorescent microangiography and confocal images were captured byoptical sectioning as described.

(h) Arteriolar Muscularization

The degree of muscularization of small arterioles was assessed in 5 μmlung cryosections immunostained for von Willebrand factor (vWF) andα-smooth muscle actin (α-SM-actin) as described in the online datasupplement.

(i) Statistical Analysis

Data was presented as mean±SEM. Differences between groups were assessedby using analysis of variance (ANOVA), followed by post hoc comparisonsusing an unpaired t test as appropriate. Differences within groupsbetween the 21- and 35-day time points were assessed using a paired ttest. Significance of differences for survival data were determinedusing the Kaplan-Meier analysis. A value of P<0.05 was consideredstatistically significant.

Results (a) In Vitro Characterization of ELPCs

After 7 to 10 days of culture in endothelial growth medium, BM-MNCsdemonstrated a cobblestone appearance typical for endothelial cells andexhibited positivity for a panel of EC markers, including Dil acLDL,UEA-1 lectin staining, and immunostaining for vWF, and Flk-1 varyingfrom 65% to 83%. ELPC phenotype was characterized in vitro (FIG. 24, athrough d) by assessing Di acetylated LDL uptake (a) and UEA-1 lectinsurface staining (b), indirect immunofluorescence staining was performedto detect vWF (c) and Flk-1 (d) expression. Fluorescently labeled ELPCswere injected 3 days after MCT administration, and lungs were perfusedwith agarose containing fluorescent microspheres just before harvest. At15 minutes after injection, cells were trapped within distal arterioles(FIG. 24, e). At later time points, injected cells were seen to engraftinto the endothelial layer of distal precapillary arterioles asconfirmed by fluorescent microangiography (FIG. 24 f). In some areas,complete luminal incorporation was observed (FIG. 24, g). Labeled ELPCsdelivered 21 days after MCT appeared to be incorporating in precapillaryand larger arterioles (FIG. 24 h and i) (vWF immunostaining; calibrationbars=50 μm).

(b) Engraftment of Fluorescently-Labeled ELPCs

CMTMR-labeled ELPCs were injected into the pulmonary circulation 3 daysafter administration of MCT. Fifteen minutes after delivery, labeledELPCs were seen distributed throughout the lung (FIG. 24( e) insert),nearly exclusively within small precapillary arterioles. Seven daysafter cell injection, fluorescently-tagged ELPCs were seen surroundingand engrafting distal arterioles (FIG. 24 f) and on occasionsintegrating into, and regenerating, the endothelium of larger arterioles(FIG. 24 g). After the first 3 days, the number of engrafted ELPCs wasfairly constant up to 3 weeks (data not shown). Similar results wereobtained when labeled ELPCs were delivered 21 days after MCI (FIGS. 24 hand 24 i).

(c) ELPC Administration in the Prevention Model

RVSP was significantly increased at day 21 after MCT compared withsaline-treated control rats (48±3 versus 26±0.9 mm Hg, P<0.001; FIG.25(A) (*P<0.001 v. control; +P<0.001 v.<CT). Administration of somaticcells (ie, skin FBs) had no protective effect (RVSP 51±5 mm Hg), whereasthe delivery of syngeneic ELPCs nearly completely prevented the rise inpulmonary systolic pressures at 3 weeks after MCT (32±1 mm Hg, P<0.001versus MCT alone). Similarly, right ventricular hypertrophy as measuredby the ratio of RV/LV weight ratio was increased in animals receivingMCT alone (0.36±0.02) or MCT with FBs (0.30±0.01; FIG. 25B). Incontrast, the delivery of bone marrow-derived ELPCs significantlyreduced right ventricular hypertrophy (0.26±0.013, P<0.01 versus MCT) toa level not significantly different from saline-treated control animals(0.23±0.01).

(d) Persistence of Protective Effects of ELPCs

FIG. 26 shows persistence of effect as examined in a separate preventionstudy over a longer time period. RVSP was measured and animalseuthanized on the development of predetermined signs of significantmorbidity. ELPC group (n=12) exhibited near normal levels of RVSP ateuthanasia with only one outlier; in contrast, RVSP in rats injectedwith fibroblasts (n=13) was markedly elevated (A, P=0.001, vs ELPCgroup). When analyzed according to time of euthanasia, there was notrend toward an increased RVSP in the ELPC-treated group even at morethan 60 days (B). Kidneys (C) from MCT-ELPC-treated rats euthanized atlater time points were enlarged with an irregular capsular surface (b),a marked contrast to those from control rats (a).

Histological examination of kidneys from MCT-ELPC-treated rats (d)revealed changes consistent with end stage renal disease includingglomerular enlargement, mesangial cell loss, and tubule hyalinization.Normal kidney histology is shown for comparison (c). In contrast to thelung (e), no fluorescently-labeled ELPCs were seen in the kidneys of MCTtreated rats 20 minutes after injection (f) (calibration bars=100 μm) orat later time points (data not shown). Glomerular enlargement, mesangialcell loss and tubule hyalinization were caused by the MCT treatment.

At the time of euthanasia, RVSP was again markedly elevated in animalsreceiving MCT alone (FIG. 26A), whereas with the exception of oneanimal, ELPC-treated rats exhibited near normal values of pulmonarysystolic pressure. Moreover, there was no tendency for RVSP to increasein the ELPC treated group over more than 60 days (FIG. 26B). Necropsystudies in the longest surviving MCI-treated rats revealed markedlyenlarged kidneys that exhibited severe histological abnormalities ofglomerular and tubular structure consistent with end-stage renal diseasein both groups (FIG. 26Ca through 26Cd). Unlike the lung, no renalengraftment of ELPCs was seen either immediately or 3 days after celldelivery (FIG. 26Ce and 26Cf).

(e) Effect of ELPC Transplantation in the Reversal Model

At 3 weeks after MCT injection, animals assigned to the three treatmentgroups exhibited similar increases in RVSP compared with saline controls(FIG. 27A) and comparable to that of the MCT alone group in theprevention protocol. Two weeks later, RVSP had progressed in the MCTanimals treated with saline. The delivery of nontransduced ELPCsprevented the further progression of PAH from day 21 (43±4 mm Hg) to day35 (36±4 mm Hg); however, only animals receiving eNOS-transduced ELPCsdemonstrated significant improvement in RVSP at day 35 (31±2 mm Hg)compared with day 21 (50±3 mm Hg, P<0.005; FIG. 28A). Of note, transgeneexpression was transient and persisted for only 1 week afterelectroporation (data not shown). The ratio of RV to LV and septalweight was significantly increased in the MCT-treated rats receivingonly saline, whereas both ELPC-treated groups displayed significantreductions compared with control rats (FIG. 27B). Similarly, the effectsof MCT on the expression of VEGF and markers of endothelial activation(E- and P-Selectin) were normalized by administration of eNOS-transducedELPCs (data not shown).

(f) Effects of ELPCs on Fluorescent Microangiography

In normal lungs, microangiography revealed an even filling of the distalarteriolar bed with a homogeneous pattern of capillary perfusion (FIG.28Aa). Immunostaining with an antibody directed to α-SM actin showedminimal muscularization of the distal arterioles in normal lungs. Incontrast, 3 or 5 weeks after MCT-induced lung injury (FIG. 28Ab and Ad,respectively), the distal arteriolar bed showed significant narrowing ofdistal arterioles with widespread capillary occlusion and evidence ofincreased distal muscularization. In animals receiving ELPCs 3 daysafter MCT, there was a marked improvement in the appearance of the lungmicrovasculature, with reservation of arteriolar continuity and enhancedcapillary perfusion (FIG. 28Ac). When ELPCs alone were administered 3weeks after MCT, only modest improvement in capillary perfusion was seen(FIG. 28Ae) with persistent distal muscularization. Only eNOS-transducedELPCs restored a more normal appearance of the lung circulation in thereversal model (FIG. 28Af). These observations were confirmed byquantification of microvascular perfusion as shown in FIG. 28B (n=6 to7/group).

(g) Arteriolar Muscularization

In normal lungs, arterioles of <30 μm showed infrequent muscularizationwith only 14% demonstrating partial muscularization (PM) and no vesselsexhibiting full muscularization (FM). In contrast, 35 days after MCTthere was a significantly higher proportion of muscularized arterioles(FIG. 28C).

Treatment with eNOS-transduced progenitor cells reduced arteriolarmuscularization, whereas nontransduced ELPCs did not, although there wasan increase overall in nonmuscularized vessels in both EPLC-treatedgroups compared with MCT alone.

(h) Survival Analysis

In one experiment, only one animal in the MCT-saline group survived tothe predefined study end point at 35 days after MCT, and therefore,these data were used for survival analysis only (FIG. 29). The injectionof ELPCs transduced with eNOS nearly completely prevented MCT-inducedmortality with all but one animal surviving to end-study (P=0.02 versusMCT alone), whereas delivery of nontransduced ELPCs produced anintermediate survival that was not significantly different from MCTalone. However, when the survival analysis was performed including all63 animals randomized in the reversal protocol, this trend persistedwith the survival benefit in MCT-treated animals receiving nontransducedELPC now reaching statistical significance (P=0.037 versus MCT alone).

DISCUSSION

Bone marrow-derived ELPCs engrafted the MCT-injured lung andincorporated into the pulmonary microvasculature, resulting in nearcomplete prevention of PAH when delivered into the pulmonary circulationwithin 3 days of MCT injury. Nontransduced ELPCs also prevented furtherincreases in RVSP when injected 3 weeks after MCI-induced lung injury.eNOS-transduced cells also resulted in normalization of pulmonaryhemodynamics in animals with established PAH, and this effect wasassociated with a significant survival benefit. Progenitor cell therapyalso resulted in marked improvement in the pulmonary microvasculararchitecture and alveolar capillary perfusion in MCT-treated animals,which could in part be attributed to repair and regeneration of lungmicrovascular endothelium.

Of interest, the protective effect of ELPCs appeared to persist as longas it was possible to follow the animals. Indeed, the prevention of MCTinduced vascular damage in the lung unmasked profound renal toxicity,which had been previously recognized in the reports that firstcharacterized the toxicity of MCT. This additional toxic effect of MCTclearly represents a significant limitation for studies of long-termsurvival after lung-specific therapy of PAH in this model.

The present results show for the first time that both ELPCs andeNOS-transduced ELPCs can dramatically improve pulmonary hemodynamicsand survival in animals with established PAH, while restoring thecontinuity of the distal arteriolar bed. These data have importantimplications for the therapy of this lethal disease and support theexploration of regenerative cell-based gene strategies for the treatmentof patients with severe refractory PAH for whom therapeutic options arevery limited and the prognosis is poor.

EXAMPLE 22 Prevention of LPS-Induced Acute Lung Injury by MesenchymalStem Cells (MSCS) Overexpressing Angipoietin-1 (ANG-1) Objectives:

The objective was to test the efficacy of MSC cells, and cell-based genetransfer using Ang-1-transduced MSC cells, to reverse or prevent AcuteRespiratory Distress Syndrome (ARDS), the clinical correlate of severeAcute Lung Injury.

Methods: Results Characterization and Ang1 Transfection of MSCs

(a) Murine MSCs were Obtained from Tulane Center for Gene Therapy (NewOrleans, La.) and Demonstrated to Differentiate into Three PredominantMesenchymal Lineages: Adipocytes, Osteocytes, and Chodrocytes (Resultsnot Shown).

The full-length coding sequence of Ang1 (1115-bp) was cloned into theexpression vector pFLAG-CMV-1 (Sigma, St. Louis, Mo.) as previouslydescribed (51). Nuclear-targeting electroporation (Amaxa, Gaithersburg,Md.) was used to transfect MSCs with Ang1 plasmid or empty vectorplasmid. Human Ang1 protein expression was verified by ELISA (R&DSystems, Minneapolis, Minn.).

Twenty-four hours after nucleofection with human plasmid Ang1 (pAng1),Ang1 protein (724±283 μg/mL) was detected in the culture supernatant(from 5×10⁵ cells), and levels were sustained for more than 5 days.Phosphorylation of Tie-2 receptor, mediated by human Ang1 (hAng1)protein expressed using the same plasmid, has been previously validatedby our group (51). Nontransfected or null-transfected (empty factor)MSCs produced no detectable Ang1 protein.

(b) Effect of MSCs Alone or Transfected with hAng1 (MSCs-pAng1) on AcuteLPS-Induced Pulmonary Inflammation

(Lipopolysaccharide)-induced pulmonary inflammation is a well known andwell documented animal model for ARDS. Measures of extent ofinflammation include cell counts from bronchoalveolar lavage (BAL) and ameasure of pro-inflammatory cytokine levels in BAL fluid and lungparenchymal homogenates. LPS-induced permeability in the lung (i.e.extent of Acute Lung Injury) can also be measured.

LPS was administered to adult C57Bl/6J mice via intratrachealinstillation, followed 30 minutes later by an injection of saline, MSCsalone, or MSCs transfected with pFLAG (null) or hAng1 plasmid(MSCs-pAng1) via the right jugular vein (FIG. 1) Human Ang1 proteinexpression was confirmed by ELISA for each batch of MSCs-pAng1 employed.Naive mice (without LPS instillation) were injected with saline or MSCsto serve as controls for any inflammatory response that might resultfrom the injected MSCs. Three days later, bronchoalveolar lavage (BAL)fluid was collected The total inflammatory cell count in the BAL fluidwas increased approximately 20-fold at day 3 following administration ofLPS. Treatment of animals with MSCs alone significantly reduced thetotal cell count in SAL fluid (FIG. 30A, p<0.05 compared to LPS/salinegroup) Treatment with MSCs-pAng1 further reduced the BAL fluid cellcount to a level similar to control, naive mice (p<0.01 for total cellscompared to LPS/saline group) Substitution of skin fibroblasts for MSCsdid not prevent the observed LPS-induced increase in BAL fluid cellcounts (data not shown).

Histological assessment of lung sections 3 days after the administrationof LPS revealed evidence of marked inflammatory infiltrates,interalveolar septal thickening, and interstitial edema (FIG. 30B). MSCsalone reduced airspace inflammation, which was more apparent in micetreated with MSCs-pAng1. Morphometric analysis measuring interalveolarseptal thickness showed a modest increase in LPS alone group, with asignificant reduction in animals receiving MSCs (data not shown).Severity of lung injury were also scored using a quantitativehistopathology score system, which evaluates lung injury in fourcategories: alveolar septae, alveolar hemorrhage, intra alveolar fibrin,and intra-alveolar infiltrates. Treatment with MSCs or MSCs-pAng1 againexhibited trends of reduced lung injury scores.

To further evaluate the anti-inflammatory actions by MSCs andMSCs-pAng1, levels of pro-inflammatory cytokines and chemokines weremeasured in BAL fluid collected from animals. Pro-inflammatory cytokines(IFN-γ, TNF-α, IL-6, and IL-1β) were all elevated in BAL fluid inresponse to LPS challenge compared with naive animals receiving saline(FIG. 31). Treatment with MSCs alone decreased the levels ofpro-inflammatory cytokines, while treatment with MSCs-pAng1 dramaticallyreduced cytokine levels to baseline values observed in naive mice. LPSinstillation also increased the levels of MIP-2, JE (murine homologue ofMCP-1), and KC (murine homologue of IL-8) in BAL fluid, whereastreatment with MSCs, and to a greater extent MSCs-pAng1, attenuatedthese increases. Similarly, LPS-induced cytokine and chemokine levels inwhole lung homogenates were significantly reduced by treatment with MSCsor MSCs pAng1 (FIG. 32), with treatment with MSCs alone as effective asMSCs-pAng1 in reducing pro-inflammatory cytokine and chemokine levels.No detectable differences in cytokine and chemokine levels in plasma wasobserved among different treatment groups, suggesting that intratrachealLPS instillation induced localized inflammation in the lungs, and didnot result in systemic inflammation in our model (data not shown).

(c) Effect of MSCs and MSCs-pAng1 on LPS-Induced ALI

Concentrations of total protein, albumin, and 1 gM were assayed oncollected BAL fluid to evaluate the integrity of the alveolar-capillarymembrane barrier and assess pulmonary vascular leakage as a marker forALI. These parameters of vascular leakage were markedly increased (totalprotein: 3-fold increase; albumin: 4-fold increase; and IgM: 25-foldincrease) in BAL fluid 3 days after LPS instillation compared to naivemice. Treatment with MSCs alone reduced total protein, albumin and IgMlevels modestly (FIG. 33A to C). Treatment with MSCs-pAng1 restoredtotal protein, albumin and IgM levels to levels not different from naïvecontrol mice (p<0.0 l for total proteins and albumin, and p<0.05 for IgMcompared to LPS/saline group, respectively).

(d) Retention and Persistence of MSCs in Mice with or withoutLPS-Induced ALI

Retention of MSCs in the lung after central venous injection wasverified by confocal microscopy and flow cytometry. MSCs labeled withthe green fluorescent cell tracker CFDA SE were observed in lungsections from both naive and LPS-challenged mice sacrificed at 15minutes (initial retention, FIG. 34A). Although labelled cells couldstill be detected 3 days after injection, they were far less abundant(FIG. 34C). No cell-specific green fluorescence was observed in sectionsfrom animals that did not receive CFDA SE-labelled cells (figure notshown). To confirm that the green fluorescence observed was indeed froman intact cell, a z-series using laser scanning confocal microscopy wasperformed showing blue nuclear staining by TO-PRO-3 surrounded by greenfluorescence by CFDA SE labelling (FIG. 34B). The percentage of theinjected MSCs retained in the lungs was quantified by flow cytometryfollowing Dispase lung digestion. An average 47% of injected cells werefound in the lungs shortly after MSCs delivery in LPS-challenged micelungs comparing to 38% in naive mice; though the difference was notstatistically significant. Regardless of lung injury, the majority ofMSCs were lost from the lung after 3 days, leaving less than 8% of cellsremaining (FIG. 34D).

CONCLUSION

MSCs alone are an effective treatment for acute lung injury, which is animportant underlying cause of human ARDS. As such, administration ofMSCs alone should be an effective treatment for ARDS. MSCs transformedto express Ang-1 before administration are an even more effectivetreatment for ARDS, resulting in near complete prevention of anyevidence of acute lung injury in response to LPS.

EXAMPLE 23 Treatment of ARDS with Skin Fibroblast Cells Expressing Ang-1(a) Cell Transfection

Skin fibroblast cells were isolated from syngeneic male Fisher344 rats(Charles River Co., Quebec, Canada) and transfected with null plasmidvector, pFLAG-CMV-1 (pFLAG), or the same vector containing thefull-length cDNA for human Ang-1 (pAng-1) using Superfect (Qiagen,Valencia, Calif.). After 24 hours, cells were suspended in Dulbecco'sPBS (Invitrogen, Carlsbad, Calif.) for injection into the pulmonarycirculation.

(b) Rat Model of ALI

All animal studies were conducted under protocols approved by the animalcare committee at St. Michael's Hospital and in accordance with CanadianCouncil of Animal Care guidelines. Male Fisher344 rats were randomlyassigned to one of four experimental groups. Transfected fibroblasts(1.5×10⁶ cells in 1 mL) were injected into the left exterior jugularvein; and 24 hours later, rats received intratracheal instillation ofsaline or LPS. Six hours after instillation, rats were tracheotomizedand bronchoalveolar lavage performed. Lavage total cells were countedusing a hemocytometer, and differential was determined by Hematoxylinand Eosin staining. Lung tissue was flash frozen and blood plasmaisolated. In separate rats, lung tissue was harvested byparaformaldehyde-inflation.

(c) Mouse Model of ALI

Female Tie2 haploinsufficient and Ang-1 overexpressing transgenic micewere randomly assigned to naive or LPS-challenged groups. Wildtypelittermates were used as controls. Mice received intratrachealinstillation of LPS and after 6 hours, bronchoalveolar lavage wasperformed and blood plasma isolated. The left lung was flash frozen andthe right lung was digested for flow cytometry. In separate mice, lungtissue was harvested by paraformaldehyde-inflation.

(d) Flow Cytometry

The right lung was perfused with 10 mL heparinized saline and inflatedwith disapaseII followed by a 1% low temperature agarose. The chestcavity was packed with ice for 5 minutes. The lung was removed andincubated in 1 mL of disapassII at room temperature for 45 minutes andthen transferred to 7 mL of DMEM with 100 U/mL DNaseI and separated intosingle cells by passing through a 70 μm cell strainer. The isolatedcells were suspended in staining buffer (2% heat-inactivated Fetal CalfSerum, 0.09% sodium azide in Dulbecco's PBS and blocked using purifiedrat anti-mouse Fc block. Cells were stained with PE-conjugated anti-CD31and one of the following biotin-conjugated antibodies: anti-E Selectin,anti-P Selectin, anti-ICAM-1, or anti-VCAM-1, with streptavidin-APC Cy7as the secondary stain. Isotype controls were used to determinebackground staining. Flow cytometry was performed using the BectonCoulter Cytomics FC500.

(e) Quantitative RT-PCR

Total RNA was extracted from lungs using TRIzol reagent and reversetranscribed. Quantitative PCR was performed using SYBR Green′ PCR MasterMix and the ABI PRISIM 79001-1T. Delta C_(T) analysis was used tocalculate expression in comparison to 18S RNA. Primers for genes ofinterest are listed in Table 1.

TABLE 1 Primers for quantitative real-time RT-PCR sense primerantisense primer Total Ang-I 5′-GAGCTCCTTGAGAATTACCTTGTGG-3'5′-CGAGTTGATTTAGTACCTGGGTCTC-3' Human Ang-I5′-CTCCAATACTCACCCTGTTATGTC-3' 5′-GACACTGGAACAGTGTGAATCTGG-3' Tie25′-AGAACAACATAGGATCAAGCAACCC-3' 5′-CTCTTCAGTTGCAACATAATCAGAAACG-3'Rat ICAM-I 5′-CAOTGCTGTACCATGATCAGAATAC-3 5′-GTAATAGGTGTAAATGGACACCAC-3'Rat VCAM-I 5′-ACGAGTGTGAATCGAAAACCGAAG-3'5′-GTATTACCAAGGAGGATGCAAAGTAG-3' Rat E-Selectin5′-GTGAGTATTCACCCTCTAATAGATG-3' 5′-CTCTCTAGAACTTGTGAACCAGAAC-3'Rat P-Selectin 5′-AGTCTTCACGAACGCTGCATATGAC-3'5′-OACCAGGAAACTTGTTATCTGCATG-3' Rat iNOS 5′-GAGACGTTCGATGTTCGAAGCAAAC-3'5′-GCTTTGTTAGGTCTAGAGACTCTG-3' Rat eNOS 5′-CTACGAAGAATGGAAGTGGTTCC-3'5′-GTGCTGAGCTGACAGAGTCGTACC-3' Rat HO-I 5′-GAAGAGGAGATAGAGCGAAACAAGC-3'5′-CTCGTGGAGACGCTTTACGTAGTGC-3' Rat ET-I 5′-GCTTCTACAGTTTCTTGTTCAGAC-3'5′-GGATGCAAACGAAGACAGGTTAGG-3' Rat Ang-2 5′-TTTGTCTCCCAGCTGACCAGTGG-3'5′-GACAGGTAGAAGTGCTCATACAG-3' Rat VEGF 5′-CATAGGAGAGATGAGCTTCCTGC-3'5′-CTCTGAACAAGGCTCACAGTGATITTC-3' I8S 5′GACGATCAGATACCGTCGTAGTTC-3'5′-GTTTCAGCTTTGCAACCATACTCC-3'

(f) ELISAs

Rat TNF-α, IL-1β and IL-6 ELISA kits and mouse TNF-α, IL-1β and IL-6,soluble ICAM-1, soluble VCAM-1, soluble E-Selectin and solubleP-Selectin ELISA kits were used following the manufacturer'sinstructions. The ET-1 ELISA kit was used following the manufacturer'sinstructions on precipitated lavage fluid spun at 3000 g for 20 minutes.

(g) Western Blots

Western Blots were performed using the following primary antibodies:rabbit anti-Tie2, goat anti-Ang-1, mouse anti-β-actin; rabbitanti-Tyr992-phospho-Tie2; goat anti-rat E-Selectin, mouse anti-rateICAM-1; and mouse anti-rat VCAM-1. Tie2 was immunoprecipitated usingrabbit anti-Tie2 antibody and membranes probed with mouseanti-phosphotyrosine (1:4000, Upstate Biotechnology; Lake Placid, N.Y.).Predetermined molecular mass standards were use as markers (Invitrogen,Carlsbad, Calif.).

(h) Immunohistochemistry and Histopathology

In separate experiments, 1.5×10⁶ CMTMR-labeled rat fibroblast cells wereinjected into naive rats, which were sacrificed 15 minutes, 24 hours or48 hours later. Confocal immunohistochemistry was performed on 15 μmlung cryo-sections stained with rabbit anti-von Willebrand Factor ormouse anti-human Ang-1.

Mouse and rat lung samples were fixed in 4% paraformaldehyde, paraffinembedded, cut into 5 μm sections, and stained with Hematoxylin andEosin. Intra-alveolar septal thickness was quantified by measuring allseptae along a crosshair placed on each image (approximately 50 septaeper animal) using ImageJ software (National Institutes of Health).

(i) Statistics

Data were represented as mean±standard error of the mean. Differencesbetween groups were assessed using ANOVA (with post hoc comparisonsusing Student-Newman-Keuls test). A value of p<0.05 was consideredstatistically significant.

Results (a) Role of Angiopoietin-1 in a Rat Model of ALI

Immunofluorescent staining of the lung with von Willebrand Factor (vWF)to label the endothelium is shown in FIG. 35A, panels a and b. Fifteenminutes after injection, CMTMR-labeled fibroblasts were visible in ornear small arterioles (panel a), persisting in the lungs 48 house later(panel b). CMTMR-labeled fibroblasts were not detectable in othertissues and have previously been shown to persist in the lung for up to6 months. Immunofluorescent staining of the lung for Ang-1 was shown inFIG. 34A, panels c and d. The expression of the Ang-1 transgene bytransplanted fibroblasts was evident by the yellow color indicatingcolocalization of CMTMR and Ang-1 signals 15 minutes after injection(panel c), again persisting for 48 hours after injection (panel d).Immunohistochemical staining confirmed that Ang-1 expressing fibroblastspersist in the lung even after LPS exposure (data not shown).Quantitative real-time RT-PCR analysis of total Ang-1 mRNA levels showeda reduction in total Ang-1 mRNA of 53% following LPS exposure comparedto saline with pFLAG-transfected cells (FIG. 35B) Pretreatment withpAng-1-transfected cells restored total Ang-1 mRNA to sham levels. Incontrast, plasmid-derived Ang-1 mRNA was undetectable in animalspretreated with pFLAG-transfected cells, while plasmid Ang-1 transcriptlevels were similar in both groups that received injection of pAng-1transfected cells (FIG. 35C). RT-PCR analysis of the receptor tyrosinekinase Tie2 demonstrated a reduction in Tie2 mRNA of 59% following LPSexposure compared to saline with pFLAG-transfected cells (FIG. 35D); andpretreatment with pAng-1-transfected cells partially restored Tie2expression. Moreover, both total Tie2 and phosphorylated Tie2 proteinwere decreased following LPS exposure compared to saline withpFLAG-transfected cells (FIG. 35E), which again was partially restoredby pretreatment with pAng-1-transfected cells.

Thus, decreases in Tie2 activity may contribute to endothelialinflammation; strategies to counteract this, such as treatment withAng-1 expressing fibroblasts may be therapeutic.

(b) Angiopoietin-1 Cell Therapy Attenuated Intra-Alveolar SeptalThickness and Airspace Inflammation in Rats:

Administration of pAng-1, compared to pFLAG, transfected cells did notsignificantly alter lung morphology in rats that received intratrachealinstillation of normal saline (FIG. 36A, panels a and b). Intra-alveolarseptal thickness and total cell count in bronchoalveolar lavage fluid(BALF) were increased 2 and 4-fold, respectively, following LPS exposurecompared to saline (FIG. 36A, panel c, FIGS. 36B and 36C). Pretreatmentof pAng-1-transfected cells significantly reduced these indicators ofvascular inflammation (FIG. 2A, panel d, FIGS. 36B and 36C). Thereduction in total BALF cells was mainly attributable to a reduction inthe number of neutrophils (Table 2). Total protein in BALF and lung wetweight to body weight ratio were increased following LPS exposurecompared to saline (FIGS. 36D and 36E). There was a trend towards areduction in these indicators of pulmonary vascular leak (p=0.10).

TABLE 2 Effect of Ang-1 cell therapy on inflammatory cells in BALFpFLAG-FB pAng-1-FB pFLAG-FB PANG-1-FB Saline Saline LPS LPS Total Cells2.5 ± 0.6 2.4 ± 0.3 10.5 ± 1.7* 6.0 ± 0.7*^(#) (10⁶ cells/mL)Neutrophils 1.9 ± 0.4 2.0 ± 0.3 10.1 ± 1.6* 5.6 ± 0.8*^(#) (10⁶cells/mL) Monocytes 5.5 ± 2.9 4.0 ± 1.0 27.5 ± 4.7* 9.5 ± 2.0*^(#) (10⁴cells/mL) Macrophages 5.6 ± 1.9 3.4 ± 0.7 1.6 ± 0.3 2.5 ± 1.6    (10⁴cells/mL) *= p < 0.05 vs. pFLAG-transfected fibroblast injected ratschallenged with saline; ^(#)= p < 0.05 vs. pFLAG-transfected fibroblastinjected rats challenged with LPS.

(c) Selective Effect of Angiopoietin-1 Cell Therapy on EndothelialAdhesion Molecule Expression in Rats:

Endothelial adhesion molecule expression is a marker of injury andactivation of endothelial cells. In animals receiving pFLAG-transfectedcells, mRNA levels for ICAM-1, VCAM-1, P-Selectin and E-Selectin inwhole lung tissue were increased by 2 to 32-fold following exposure toLSP compared to saline (FIG. 37A-D). the increases in ICAM-1, VCAM-1 andP-Selectin mRNA levels following LPS were similar in rats receivingpang-1 compared to pFLAG-transfected cells. In contrast, pretreatmentwith pAng-1-transfected cells largely prevented the increase in mRNAexpression of the endothelial-selective adhesion molecule, E-Selectin,in LPS treated rats (FIG. 37C). Western blot analysis showed nodifference in ICAM-1 or VCAM-1 protein expression between theexperimental groups, whereas E-Selectin protein was induced by LPSexposure and this was substantially reduced by the administration ofp-Ang-transfected cells.

EXAMPLE 24 Role of Angiopoietin-1 in Transgenic Models of ALI

Experiments were performed using Tie2 heterozygous deficient mice(Tie2+/−) or binary transgenic mice in which Dox-conditional Ang-1overexpression was targeted to endothelium using the Tie1 promoter(tTA-Ang-1). Wildtype (WT) littermates were used as controls. Total lungAng-1 protein expression by Western Blot analysis was not differentbetween endothelial-targeted, Ang-1 overexpressing mice and WT or Tie2heterozygous deficient mice (FIG. 38A). This may be a result of highbasal levels of pulmonary Ang-1 expression in extra-endothelial cellsunder normal conditions, which may overshadow the human Ang-1 transgeneexpression which is restricted to the endothelium. However, the decreasein Ang-1 protein expression seen in WT mice following LPS challenge wasblunted in tTA-Ang-1 mice. Paradoxically, the LPS-induced reduction inAng-1 was also attenuated in Tie2 deficient mice, which we hypothesizemight reflect a compensatory mechanism for the very low expression ofTie2 in these animals. As expected, basal Tie2 expression was reduced byabout 50% in Tie2+/− compared with WT mice (FIG. 38B) and Tie2 proteinwas markedly downregulated by LPS exposure in WT mice, which was furtherreduced in Tie2+−.

(a) Septal Thickness and Airspace Inflammation in Transgenic Mice:

In the absence of LPS, lung morphology was not significantly differentbetween groups of transgenic mice (FIG. 39A). The intra-alveolar septalthickness increased 1.7-fold following LSP exposure in WT mice (FIG.39B). This increase in septal thickness was significantly blunted inAng-1 overexpressing mice, whereas Tie2 deficient mice exhibited nearlya 50% greater increase in intra-alveolar septal thickness in response toLPS compared to WT animals. LPS-induced airspace inflammation mirroredthese differences, with the increase in total cells in BALF,attributable mainly to an increase in neutrophils, being markedlyblunted in Ang-1 overexpressing mice, whereas Tie2 deficient animalsconsistently exhibited exaggerated air space inflammation (FIG. 39C,Table 5). Total protein in BALF was increased 3-fold following LPS in WTmice and again this was suppressed in Ang-1 overexpressing mice andincreased in Tie2 deficient animals (FIG. 39D). In addition, mortalitywas higher in Tie2 haploinsufficient mice subjected to LPS challenge(60% mortality within 1 hour) compared to the other experimental groups(17%; p<0.02). Necropsy revealed that mortality was associated withmassive alveolar flooding in Tie2 deficient mice.

TABLE 5 Inflammatory cells in BALF of transgenic mice. WT Ang-1-tTA Tie2+/− WT Ang-1-tTA Tie2 +/− Naïve Naïve Naïve LPS LPS LPS Total Cells 1.7± 0.3 1.6 ± 0.1 2.6 ± 1.1 19.9 ± 1.4*   6.3 ± 1.4*^(#) 44.4 ± 8.6* (10⁶cells/mL) Neutrophils 5.6 ± 0.9 2.3 ± 0.5 3.3 ± 0.9 164 ± 13*  47.1 ±6.4*^(#)  345 ± 17*^(#) (10⁵ cells/mL) Monocytes 0.3 ± 0.2 0.0 ± 0.0 0.5± 0.3 16.0 ± 3.6*   1.8 ± 0.5*^(#)  24.9 ± 6.5*^(#) (10⁵ cells/mL)Macrophages 1.1 ± 0.1  1.3 ± 0.05  2.4 ± 0.1* 1.9 ± 0.9 1.4 ± 0.67.42.0*^(#) (10⁵ cells/mL) *= p < 0.05 vs. WT naive mice; ^(#)= p < 0.05vs. WT mice challenged with LPS

(b) Ang-1 Overexpression Reduces Endothelial Expression of AdhesionMolecules:

To evaluate changes in the expression of adhesion molecules inendothelial cells versus other lung cell types, mouse lungs weredigested in dispase and stained for the endothelial cell marker, CD31.Subsequently, flow cytometry was performed for each adhesion moleculeseparately using 2-colour flow cytometry, gated around the CD31+ andCD31− populations. The percentage of endothelial cells positive forE-Selectin, P-Selectin, ICAM-1 and VCAM-1 increased from 4 to 34-foldfollowing LPS in wildtype mice (FIG. 40A, left hand panels).Endothelial-targeted Ang-1 overexpression nearly completely suppressedthe LPS-induced increases in endothelial expression of E-Selectin,P-Selectin and VCAM-1 (FIGS. 40A, B and D), but had only a marginaleffect on ICAM-1 expression (FIG. 40C). Surprisingly, Tie2 deficientmice showed similar, although more modest, reductions in adhesionmolecule expression in response to LPS. This may be explained by greateradhesion molecule shedding in these animals (FIG. 41), consistent withmore advanced inflammation. As well, similar changes, though with loweroverall numbers of positive cells, were seen when the expression ofthese adhesion molecules was studied in all dispersed lung cells (FIG.40, right hand panels).

DISCUSSION

In both the rat and mouse models, Ang-1 overexpression markedly reducedseptal edema in response to LPS, as well as alveolar and endothelialinflammation. This data supports the potential utility of Ang-1 in thetreatment of acute lung injury.

The endothelial monolayer plays a critical role in many aspects of thepathogenesis of ALI and ARADS. Alterations in the production ofvasoactive mediators by injured endothelium leads to impaired hypoxicpulmonary vasoconstriction. Increased expression of angiogenic growthfactors, such as VEGF, contributes to increased endothelial permeabilityand interstitial edema, increased pulmonary dead space, and vascularremodeling. However, perhaps the most important role of the endotheliumin ALI and ARDS is in regulation of inflammation. Leukocyte adhesion tothe endothelium is a prerequisite for migration into the lungparenchyma, where the inflammatory cells contribute to lung injury.Indeed, previous studies have reported attenuation of lung injury inexperimental ALI by blocking endothelial adhesion molecules. Thus,strategies to selectively reduce endothelial inflammation in response toinjury could be of potential benefit, not only in ALI and ARDS, but alsoin systemic inflammatory disorders such as the Systemic InflammatoryResponse Syndrome, sepsis and the Multiple Organ Dysfunction/FailureSyndrome.

Ang-1 cell-based gene transfer also resulted in several downstreameffects in the rat model of ALI. Expression of two protective enzymes,eNOS and HO-1, was increased following Ang-1 cell therapy inLPS-challenged rats. Mice overexpressing eNOS were previously shown tobe protected from lung injury during endotoxic shock. HO-1 is known toincrease during ARDS and the increase in HO-1 activity is thought to bea protective mechanism. In addition, ET-1, which was previously shown toincrease inflammation and pulmonary-vascular leaks in ARDS, was reducedfollowing Ang-1 cell therapy compared to LPS-challenged controls. ET-1receptor antagonists have been shown to be protective in an experimentalmodel of ALI, thus the mechanisms of protection of Ang-1 likely includemodulation of the production of vasoactive factors.

Ang-1 cell therapy represents a therapeutic strategy to preventdevelopment of clinical ARDS. Cell-based gene therapy could beadvantageous over intravenous administration of recombinant Ang-1protein, as it allows for more targeted expression of the transgene andovercomes issues of short protein half-life after injection. Inaddition, endothelial progenitor cells and mesenchymal stem cells mayalso provide protection above and beyond that provided by increasedAng-1 expression, as these cells may release many additional mediatorsthat could be beneficial or participate directly in the repair andregeneration of the endothelial-epitheliol barrier or exert immunemodulatory effects that reduce lung inflammation and injury.

The benefits of Ang-1 overexpression were seen in two experimentalmodels, showing reduced vascular endothelial inflammation and leak.Overexpression of Ang-1 blunted endothelial adhesion moleculeexpression, increased HO-1 and eNOS expression, and decreased ET-1expression, as well as reduced airspace inflammation, reducedintra-alveolar septal thickening and reduced early mortality. Therefore,these results show that Ang-1 therapy is a viable new treatment strategyto reduce the vascular consequences of lung injury, which are a majordeterminant of morbidity and mortality in critically ill patients.

1. A process for alleviating or inhibiting the progression ofmechanically induced acute lung injury in a mammalian patient comprisingadministration of syngeneic or allogeneic or autologous mesenchymal stemcells, smooth muscle cells, fibroblast cells or a combination thereof tothe lung by injection into the pulmonary circulation of the mammalianpatient suffering from the disorder wherein the administration resultsin alleviation or inhibition of the progression of the acute lunginjury.
 2. The process of claim 1, wherein said cells have beentransformed in vitro to express a transgene.
 3. The process of claim 1wherein the cells are skin fibroblast cells.
 4. The process of claim 1wherein the cells are allogenic.
 5. The process of claim 1 wherein thecells are autologous.
 6. The process of claim 2 wherein thetransformation is through electroporation with angiopoietin-1 clonedinto a plasmid vector.
 7. The process of claim 1, wherein the acute lunginjury is lung inflammation, septal edema, alveolar inflammation,endothelial inflammation or a combination thereof.
 8. The process ofclaim 1, wherein the acute lung injury is ARDS.
 10. The process of claim2, wherein the transgene is vascular endothelial growth factor,fibroblast growth factor, angiopoietin, hemoxygenase, transforminggrowth factor, hepatic growth factor, endothelial nitric oxide synthase,Angiopoietin-1, prostaglandin I synthase (PGIS) hypoxia induciblefactor, or a combination thereof.
 11. The process of claim 10 whereinthe transgene is endothelial nitric oxide synthase.
 12. The process ofclaim 10, wherein the transgene is vascular endothelial growth factor.13. The process of claim 10 wherein the transgene is PGIS.
 14. Theprocess of claim 10 wherein the transgene is Ang-1.
 15. The process ofclaim 1, wherein the mechanical injury is ventilation induced lunginjury.
 16. A mechanical lung injury alleviating therapeutic comprisingmesenchymal stem cells, smooth muscle cells, fibroblast cells or acombination thereof, wherein the therapeutic is delivered intraarterially or intravenously.
 17. The therapeutic of claim 16, whereinthe mesenchymal stem cells, smooth muscle cells, fibroblast cells or acombination thereof have been transformed with a transgene.
 18. Thetherapeutic of claim 17, wherein the transgene is vascular endothelialgrowth factor.
 19. The therapeutic of claim 17, wherein the transgene isAng-1.
 20. The therapeutic of claim 16, where the lung injury is ARDS.